Assessment of the Endocrine-Disrupting Effects Of

Food and Chemical Toxicology 133 (2019) 110759

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Food and Chemical Toxicology

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Assessment of the endocrine-disrupting effects of organophosphorus T pesticide triazophos and its metabolites on endocrine hormones biosynthesis, transport and receptor binding in silico ⁎ Fang-Wei Yanga, Yi-Xuan Lia, Fa-Zheng Rena,b, Jie Luoa,c, Guo-Fang Panga,d, a Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, 100083, China b Key Laboratory of Functional Dairy, Co-constructed by Ministry of Education and Beijing Government, Beijing Laboratory of Food Quality and Safety, China Agricultural University, Beijing, 100083, China c College of Food Science and Technology, Hunan Agricultural University, Changsha, 410114, China d Chinese Academy of Inspection and Quarantine, Beijing, 100176, China

ARTICLE INFO ABSTRACT

Keywords: Triazophos (TAP) was a widely used organophosphorus insecticide in developing countries. TAP could produce Triazophos specific metabolites triazophos-oxon (TAPO) and 1-phenyl-3-hydroxy-1,2,4-triazole (PHT) and non-specific Metabolites metabolites diethylthiophosphate (DETP) and diethylphosphate (DEP). The objective of this study involved Endocrine disruption computational approaches to discover potential mechanisms of molecular interaction of TAP and its major Hormones metabolites with endocrine hormone-related proteins using molecular docking in silico. We found that TAP, Molecular docking TAPO and DEP showed high binding affinity with more proteins and enzymes than PHT and DETP. TAPmight interfere with the endocrine function of the adrenal gland, and TAP might also bind strongly with glucocorticoid receptors and thyroid hormone receptors. TAPO might disrupt the normal binding of androgen receptor, estrogen receptor, progesterone receptor and adrenergic receptor to their natural hormone ligands. DEP might affect biosynthesis of steroid hormones and thyroid hormones. Meanwhile, DEP might disrupt the bindingand transport of thyroid hormones in the blood and the normal binding of thyroid hormones to their receptors. These results suggested that TAP and DEP might have endocrine disrupting activities and were potential endocrine disrupting chemicals. Our results provided further reference for the comprehensive evaluation of toxicity of organophosphorus chemicals and their metabolites.

1. Introduction represented a risk to human health as well as the ecological system due to its high chemical and photochemical stability (http://sitem.herts.ac. 1.1. Chemical characteristics, residues and toxicity of triazophos uk/aeru/iupac/Reports/653.htm). According to the classification standard of World Health Organization (WHO), TAP was a Class Ib toxic Triazophos/Triazofos/Hostathion/Phentriazophos (O,O-diethyl-O- organophosphorus insecticide (WHO, 2010). The neurotoxicity, hepa- (1-phenyl-1H-1,2,4-triazol-3-yl) phosphorothioate, CAS Registry No. totoxicity, nephrotoxicity, reproductive toxicity and genotoxicity of

24017-47-8, chemical formula C12H16N3O3PS, molar mass TAP attracted considerable public attention over the last decade. It was 313.31 g mol−1, log Kow 3.55, abbreviated as TAP) was an efficient and reported that TAP had fairly high lethal toxicity to aquatic creatures broad-spectrum organophosphorus pesticide (OP) used as insecticide, and posed a threat to the health of aquatic ecosystems (Wang et al., nematicide and acaricide, which was widely used in Asian countries, 2010; Wu et al., 2018; Zhang et al., 2018a,b,c). Recent research also such as China, India, Pakistan, to protect various crops like cotton, rice, revealed that TAP induced oxidative stress and histomorphological wheat, tea, fruits, oil seeds and vegetables (Bhandari et al., 2019; Chen changes in rats (Jain et al., 2011, 2013; Sharma and Sangha, 2014; et al., 2009; Duan et al., 2016; Fang et al., 2015; Hong et al., 2019; Sharma et al., 2015a, 2015b). Zhang et al. (2011) found that TAP Kumari and John, 2019). However, the widespread application of TAP chronic dietary intake represented a significant risk to the elderly

⁎ Corresponding author. Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science and Nutritional Engineering, China Agricultural University. No. 17 Qinghua East Road, Haidian District, Beijing, 100083, China. E-mail address: [email protected] (G.-F. Pang). https://doi.org/10.1016/j.fct.2019.110759 Received 18 June 2019; Received in revised form 31 July 2019; Accepted 12 August 2019 Available online 14 August 2019 0278-6915/ © 2019 Elsevier Ltd. All rights reserved. F.-W. Yang, et al. Food and Chemical Toxicology 133 (2019) 110759 persons and an acute nutritional intake risk of TAP residues in apple, 1.4. Screening, identification and risk assessment of endocrine disrupting cabbage, rice and wheat meal reached an unacceptable range in China. chemicals in silico Therefore, the wide application of TAP raised concerns on the environmental pollution and the potential risk to human health. Exogenous chemicals, exposed through the environmental and dietary pathways, had potential endocrine disruption effects that harm human health and the ecosystem (Giulivo et al., 2016; He et al., 2015; 1.2. Major degradation metabolites of TAP Mimoto et al., 2017; Pande et al., 2019). Endocrine disrupting chemicals (EDCs) caused a variety of diseases, such as reproductive diseases, After reaching the liver, TAP would produce specific metabolites metabolic diseases and even cancers, by interfering with the endocrine triazophos-oxon (TAPO), 1-phenyl-3-hydroxy-1,2,4-triazole (PHT), and system (The Lancet Diabetes & Endocrinology, 2019). If all the potential non-specific metabolites diethylthiophosphate (DETP) and diethylpho- EDCs in the environment and foods were to be screened via traditional sphate (DEP) under the action of a series of metabolic detoxification in vitro and in vivo methods, this would constitute an unbearable fi- enzymes (Bock and Their, 1976; Schwalbe-Fehl and Schmidt, 1986; nancial burden, would conflict with the “3R” principles of animal-use Wang et al., 2015). Briefly, TAP desulfurized under the action of ethics, and the work would stretch over many years, making it difficult CYP450 enzymes to produce triazophos-oxon (TAPO). Because the in- to meet the needs of toxicity and ecological risk assessment for EDCs hibition of acetylcholinesterase by TAPO was stronger, this process was (Burgdorf et al., 2019; Chen et al., 2018). In recent years, with the rapid actually a “metabolic increased toxification” process, but TAPO was development of computer technology and three-dimensional structure more conducive to the subsequent hydrolysis of paraoxonase 1, and analysis methods of proteins, molecular docking technology has been TAPO was degraded into PHT and DEP. TAP could also be directly gradually applied to toxicological evaluation field, and computational decomposed into PHT and DETP under the action of glutathione and toxicology research was increasing, especially the study of EDCs glutathione S-transferase, and then DETP could be further desulfurized (Browne et al., 2018; Selvaraj et al., 2018; Sun et al., 2019). Compu- into DEP. It was worth noting that DETP and DEP were non-specific tational toxicology has been recommended as the screening and pre- metabolites of many OPs, such as TAP, chlorpyrifos, diazinon, para- dicting method by the Organization for Economic Co-operation and thion, and phorate. More importantly, both DETP and DEP had high Development (OECD) (OECD, 2018), the US Environmental Protection bioavailability, and the molar mass of human exposure to DETP and Agency (EPA) (Browne et al., 2017), and others. Meanwhile, the Nobel DEP was higher than that of OPs (Forsberg et al., 2011; Sudakin and Prize in Chemistry 2013 was awarded to Martin Karplus, Michael Le- Stone, 2011; Timchalk et al., 2007; Zhang et al., 2008). In fact, ap- vitt, and Arieh Warshel for developing the multiscale models of com- proximately 75% of the commonly used OPs could be metabolized and plex chemical systems (Karplus et al., 2013), which demonstrated that degraded into six dialkyl phosphates (DAPs) in vivo and in the en- in this era of exceptional computational power, in silico analyses could vironment, namely diethyldithiophosphate (DEDTP), DETP, DEP, di- be as important as those conducted in vitro, and computer molecular methyldithiophosphate (DMDTP), dimethylthiophosphate (DMTP), and simulation methods have made significant contributions to the devel- dimethylphosphate (DMP) (Shomar et al., 2014; Ueyama et al., 2015; opment of life sciences and drug researches. Molecular docking pro- Yusa et al., 2015). The concentrations of DAPs in human urine were vided another reasonable method for the evaluation of EDCs, especially often used as the biomarkers for the exposure of OPs to assess the for some compounds with missing experimental data, the application of correlation between OPs exposure and diseases (Bernieri et al., 2019; molecular docking to explore the roles of EDCs and putative targets can Omoike et al., 2015; Panuwet et al., 2018; Shrestha et al., 2018; Wang supplement limited experimental data and improve the overall toxicity et al., 2017a,b). Interestingly, some organophosphorus flame retardants assessment of EDCs (Chen et al., 2018; Vuorinen et al., 2015; Yuriev and plasticizers could also be degraded and metabolized to DEP (Chu et al., 2015). Molecular docking or molecular simulation in silico pro- and Letcher, 2018; Li et al., 2017; Reemtsma et al., 2011; Sun et al., vided another effective way to evaluate the safety of EDCs more com- 2018). prehensively by elucidating the interaction mechanism between exogenous small molecules and biological macromolecules at the molecular level (Hazarika et al., 2019; Jeong et al., 2019). 1.3. Endocrine disrupting effects of OPs 1.5. Biosynthesis, transport, and receptors binding of endocrine hormones Many OPs have been reported to impact levels of the hormones involved in the hypothalamic-pituitary-adrenal (HPA) axis (Raees et al., Corticosteroid and catecholamine hormones were secreted by the 2012), hypothalamic-pituitary-thyroid (HPT) axis (Ahmad et al., 2018; adrenal glands, androgens and estrogens were secreted by the gonads, Akande et al., 2016; Chebab et al., 2017; Mosbah et al., 2016), and and thyroid hormones were secreted by the thyroid gland (La Perle and hypothalamic-pituitary-gonadal (HPG) axis (Geng et al., 2015; Jallouli Dintzis, 2018; Rosol et al., 2013; Wallig, 2018). It was precise because et al., 2016; Sharma et al., 2015a). Chlorpyrifos, a widely-studied OP, of the important physiological role of endocrine hormones that they was found to upset the thyroid and adrenal gland homeostasis both in were usually disturbed by EDCs such as exogenous environmental human and animal models (Chebab et al., 2017; John and Shaike, chemicals and dietary exposure pollutants, which affected the growth, 2015) and exert an inhibitory effect on sex hormones (Adedara et al., development, reproduction, and survival of humans and animals 2018; Peiris and Dhanushka, 2017). Furthermore, there were studies (Evans, 2017). Biosynthesized cholesterol through the key enzyme 3- reporting histopathological injury to the thyroid and parathyroid in hydroxy-3-methylglutaryl-CoA reductase (HMGR), which could be mice induced by diazinon (Ahmad et al., 2018), as well as the inhibition transported into the inner mitochondrial membrane by steroidogenic effect of malathion in thyroid stimulating hormone-dependent pathway acute regulatory protein (StAR), where it was cleaved by cholesterol and transcription of thyroglobulin in FRTL-5 cell line (Xiong et al., side-chain cleavage enzyme (CYP11A1) to pregnenolone, the common 2017). The effects of TAP on thyroid have only been studied inzebra- precursor for other steroid hormones (Shen et al., 2016). The adrenal fish embryos by measuring the mRNA levels of thyroid hormones (Wu cortex and gonads were the sites of steroid hormone synthesis and et al., 2018), lacking more comprehensive evidences. All these OPs different steroidogenic enzymes, such as steroid 17-alpha-hydroxylase/ were metabolized to DAPs in the environment and in vivo; however, it 17,20 lyase (CYP17A1), steroid 21-hydroxylase (CYP21A2), aldos- was unclear whether these effects were induced by the parent OPsor terone synthase (CYP11B2), aromatase (CYP19A1), and estradiol 17- the metabolites, especially the specific and the non-specific metabolites. beta-dehydrogenase 1 (17β-HSD1), which were all regulated by transcription factor steroidogenic factor 1 (SF-1), were expressed in the adrenal cortex and gonads (Shen et al., 2016). The enzymes tyrosine

2 F.-W. Yang, et al. Food and Chemical Toxicology 133 (2019) 110759

Table 1 Information for selected receptors in PDB database.

Names Abbreviation PDB entry Resolution (Å) Residue count Organism

3-hydroxy-3-methylglutaryl-CoA reductase HMGR 1DQ9 2.8 1868 Homo sapiens Steroidogenic factor 1 SF-1 4QJR 2.4 245 Homo sapiens Steroidogenic acute regulatory protein StAR 3P0L 3.4 884 Homo sapiens Cholesterol side-chain cleavage enzyme CYP11A1 3N9Z 2.17 974 Homo sapiens Aldosterone synthase CYP11B2 4DVQ 2.49 5796 Homo sapiens Steroid 17-alpha-hydroxylase/17,20 lyase CYP17A1 4NKX 2.794 1976 Homo sapiens Aromatase CYP19A1 3S79 2.75 503 Homo sapiens Steroid 21-hydroxylase CYP21A2 4Y8W 2.64 1446 Homo sapiens Estradiol 17-beta-dehydrogenase 1 17β-HSD1 6CGC 2.1 656 Homo sapiens Tyrosine hydroxylase TH 2XSN 2.68 1372 Homo sapiens Aromatic L-amino acid decarboxylase AAAD 3RCH 2.8 480 Homo sapiens Dopamine beta-hydroxylase DBH 4ZEL 2.9 1156 Homo sapiens Phenylethanolamine N-methyltransferase PNMT 4MQ4 2.2042 578 Homo sapiens Iodotyrosine deiodinase IYD 4TTB 2.447 530 Homo sapiens Human serum albumin HSA 2BX8 2.7 1170 Homo sapiens Corticosteroid binding globulin CBG 2VDY 2.3 746 Homo sapiens Sex hormone-binding globulin SHBG 1D2S 1.55 170 Homo sapiens Thyroxine-binding globulin TBG 2CEO 2.8 758 Homo sapiens Transthyretin TTR 1ICT 3.0 1016 Homo sapiens Glucocorticoid receptor GR 4MDD 2.4 516 Homo sapiens Mineralocorticoid receptor MR 2AA2 1.95 275 Homo sapiens Androgen receptor AR 2AM9 1.64 266 Homo sapiens Constitutive androstane receptor CAR 1XVP 2.6 1016 Homo sapiens Estrogen receptor alpha ERα 2IOG 1.6 246 Homo sapiens Estrogen receptor beta ERβ 1QKM 1.8 255 Homo sapiens Estrogen related receptor alpha ERRα 3K6P 1.996 248 Homo sapiens Estrogen related receptor gamma ERRγ 2P7G 2.1 251 Homo sapiens Progesterone receptor PR 1SQN 1.451 522 Homo sapiens Thyroid hormone receptor alpha TRα 4LNW 1.9 267 Homo sapiens Thyroid hormone receptor beta TRβ 3GWS 2.2 259 Homo sapiens Beta-1 adrenergic receptor β1AR 4BVN 2.1 315 Meleagris gallopavo Beta-2 adrenergic receptor β2AR 3NY9 2.84 490 Homo sapiens D2 dopamine receptor D2DR 6CM4 2.87 430 Homo sapiens D3 dopamine receptor D3DR 3PBL 2.89 962 Homo sapiens D4 dopamine receptor D4DR 5WIU 1.96 422 Homo sapiens hydroxylase (TH), aromatic L-amino acid decarboxylase (AAAD), do- family, SHBG and apolipoproteins (Benvenga and Guarneri, 2016). The pamine beta-hydroxylase (DBH), and phenylethanolamine N-methyl- relationship between hormone molecules and receptor proteins also transferase (PNMT), which were related to catecholamines biosynthesis played an important role in signal transduction of endocrine systems. in adrenal medulla (Kvetnansky et al., 2009, 2013). The iodotyrosine Hormone nuclear receptors, such as glucocorticoid receptor (GR), mi- deiodinase (IYD) salvaged iodide from mono- and diiodotyrosine neralocorticoid receptor (MR), androgen receptor (AR), constitutive formed during the biosynthesis of the thyroid hormones, and the rescue androstane receptor (CAR), estrogen receptor alpha (ERα), estrogen and recycling of iodide by the action of IYD (Targovnik et al., 2017; receptor beta (ERβ), estrogen related receptor alpha (ERRα), estrogen Thomas et al., 2009). related receptor gamma (ERRγ), progesterone receptor (PR), thyroid Binding proteins in the peripheral circulation were important in hormone receptor alpha (TRα), thyroid hormone receptor beta (TRβ), regulating the transport, bioavailability, and metabolism of their cog- belonged to a kind of superfamily hormone receptors with similar nate ligands, such as hormones, fatty acids, vitamins, and drugs structure and function. After binding with ligands, they entered the (Goldman et al., 2017). The functions of human serum albumin (HSA) nucleus and perform the function of transcription factors to activate the were the maintenance of colloid osmotic blood pressure, the transport expression of downstream target genes (Gronemeyer et al., 2004; of hormones, fatty acids, drugs, other compounds and buffering pH, and Ribeiro Filho et al., 2019; Weikum et al., 2018). Adrenergic receptors its binding also modulate acute and chronic toxicity (Grumetto et al., (α and β subtypes) were the basic targets of endogenous neuro- 2019). The major sex steroid hormones—testosterone, 5α-dihy- transmitters noradrenaline and adrenaline, especially in mediating drotestosterone, and 17β-estradiol—bound predominantly to sex hor- sympathetic activation to peripheral organs, but also in the central mone-binding globulin (SHBG) and to HSA and to a lesser extent to nervous system (Pytka et al., 2016). Dopamine belonged to catechola- corticosteroid binding globulin (CBG) (Goldman et al., 2017). CBG was mines alongside noradrenaline and adrenaline. It was not only a pre- also the primary transporter for glucocorticoids (cortisol and corticos- cursor of the latter, but also exerted its physiological effect (Pytka et al., terone) and progestins (progesterone and 17-hydroxyprogesterone), 2016). Dopamine receptors were G protein-coupled ones; they were and it regulated the partitioning of circulating cortisol into bound and expressed, inter alia, in the central nervous system, with two families unbound fractions (Bolton et al., 2014; Goldman et al., 2017). Thyr- amongst them were distinguished: D1-like comprising D1 and D5 re- oxine-binding globulin (TBG), transthyretin (TTR), HSA could bind and ceptors and D2-like consisting of D2, D3, and D4 (Beaulieu and transport thyroid hormones (Benvenga and Guarneri, 2016), and TBG Gainetdinov, 2011). had the highest affinity and carries most of thyroxine and triiodothyr- In this study, the potential endocrine disruption effects of TAP and onine in human serum, TTR and HSA bound only a small proportion of its major metabolites were evaluated using the Discovery Studio 2019 thyroxine and triiodothyronine despite large capacity (Janssen and in silico. Molecular docking in silico using Discovery Studio software has Janssen, 2017). TBG homologues were α1-antitrypsin, α1-antic- also been reported in the literatures (Cheurfa et al., 2019; da Silva hymotrypsin, antithrombin III, and CBG, while TBG nonhomologous Hage-Melim et al., 2019; Zengin et al., 2018). This study mainly as- carriers were α1-acid glycoprotein, which belonged to the lipocalin sessed the disturbance of TAP and its main metabolites on the

3 F.-W. Yang, et al. Food and Chemical Toxicology 133 (2019) 110759 physiological processes of the adrenal glands, gonads, thyroid gland methods. Among them, the CDOCKER, a CHARMm-based docking en- related hormones biosynthesis key enzymes/proteins, blood circulation gine (Wu et al., 2003), was a flexible molecular docking procedure, transport binding proteins, binding receptors and others. The crystal which could be applied to the study of receptor-ligand induced con- structures of all selected enzymes/proteins have been identified and jugation, interaction modes and related functional mechanisms, im- their ligand binding domain active sites were known. This study pro- proving the accuracy of the docking results. With CDOCKER, initial vided more detailed and comprehensive parameters for the toxicity ligand conformations were sampled via high temperature molecular assessment of TAP or other OPs with similar chemical structures and dynamics and were also allowed to flex during the refinemen. Crucially, their possible mechanisms of endocrine disruption. CDOCKER also provided a physics-based scoring function, via the CHARMm energy of the docked complex. CDOCKER has been shown to 2. Materials and methods give highly accurate docked poses (Erickson et al., 2004). Previously processed receptor proteins were defined as receptor 2.1. Receptors preparation molecules by Define and Edit Binding Site under the Receptor-Ligand Interactions tool. The location and size of the active sites were de- The structure files of proteins such as biosynthesis key enzymes/ termined according to the position of the original ligand of the receptor proteins, transport proteins, and receptors for hormones were derived protein. Using Docking Optimization under the Receptor-Ligand from the Protein Data Bank (PDB) (http://www.rcsb.org/), and see Interactions tool in Discovery Studio 2019, we opened the docking Table 1 for details. Then the Protein Clean tool under Manual Pre- process of CDOCKER, set the corresponding parameters in turn, and paration in the Macromolecules Tool in Discovery Studio 2019 (version added CHARMm force field to the ligand and receptor molecules re- 19.1.0.18287, Dassault Systèmes BIOVIA, Discovery Studio Modeling spectively for molecular docking. 178 Environment, Release 2018, San Diego, CA, USA) was used to correct the issues of non-standard named amino acids, inconsistent 2.4. Analysis of molecular docking results conformations, wrong bonds, number of hydrogen bonds, missing side chains and skeleton atoms in PDB file proteins. After cleaning the The Receptor-Ligand Interactions toolbar under Discovery Studio protein, the hydrogen bond on the protein was removed by default, so it 2019 was used to view the interactions between ligands and receptor was necessary to add hydrogen atoms manually and use the added protein complexes, such as hydrogen bonds, Pi-Pi bonds, Pi-Sulfur Hydrogens option in the Chemistry tool to add polar hydrogen to the bonds, salt bridges, and attraction charge. We could also use the receptor protein. Each receptor protein itself already contained a small Analyze Ligand Pose process in the Receptor-Ligand Interactions ligand molecule, which was located at the active sites of the protein, so toolbar of Discovery Studio 2019 to further analyze the poses obtained the small molecule ligand was deleted. by molecular docking. The docking analysis mainly included: the number of hydrogen bonds formed by the docked poses and the re- 2.2. Ligands preparation ceptor protein complex, the number of hydrogen bonds formed with each amino acid residue, the near contact with the entire complex, and The structure files and characteristic information of TAP andits the near contact with each amino acid residue etc. At the same time, major metabolites were referenced from the PubChem (https:// used the lowest CDOCKER_INTERACTION_ENERGY to evaluate con- pubchem.ncbi.nlm.nih.gov/); see Table 2 for details. Then the ligand formations during docking simulations. The lower the binding energy, molecules were further optimized using the Minimization in the Small the stronger the interactions between the ligand and receptor protein. Molecules tool in Discovery Studio 2019. 2.5. Reliability verification of molecular docking 2.3. Molecular docking using CDOCKER While the ligands such as TAP and its major metabolites were Discovery Studio 2019 provided several different molecular docking docked into receptor protein molecules, the accuracy and reliability of

Table 2 Structures and features of TAP and its major metabolites.

Compounds Structure Molecular Formula CAS No. PubChem CID

Triazophos (TAP) C12H16N3O3PS 24017-47-8 32184

Triazophos-oxon (TAPO) C12H16N3O4P – –

1-phenyl-3-hydroxy-1,2,4-triazole (PHT) C8H7N3O 4231-68-9 77910

Diethyl thiophosphate (DETP) C4H11O3PS 2465-65-8 655

Diethyl phosphate (DEP) C4H11O4P 598-02-7 654

4 F.-W. Yang, et al. Food and Chemical Toxicology 133 (2019) 110759 molecular docking need to be investigated. All of the receptor proteins Table 4 selected in this study contained the original ligands. The original li- The lowest interaction energy (kcal/mol) between TAP, TAP's major metabo- gands were deleted first, and then docked to the original active sites. lites and steroid hormones biosynthesis-related enzymes/proteins. The root mean squared deviation (RMSD) values of the conformations Receptors TAP TAPO PHT DETP DEP of the original ligands and TAP and its major metabolites were then compared. Since the resolution of the crystal structure in the PDB da- HMGR −32.5477 −37.0916 −22.1141 −25.1874 −31.2638 tabase was approximately 2 Å, we believed that there was no difference SF-1 −28.8641 −28.9289 −17.8068 −18.2077 −32.8502 StAR −34.1590 −37.7853 −23.6999 −23.7911 −23.8388 in the two molecular structures when the RMSD was within 2 Å. If the CYP11A1 −29.3898 −32.2015 −23.0100 −20.9167 −39.8313 RMSD < 2.0 Å, the reliability of the docking method and the rationality CYP11B2 −31.5445 −37.5892 −18.9738 −16.5541 −49.5906 of the docking parameters could be confirmed, and the strong interac- CYP17A1 −36.1763 −35.8308 −21.9371 −22.1311 −23.1907 tion between TAP and its major metabolites and receptor proteins could CYP19A1 −34.5617 −38.8542 −24.3682 −19.9855 −43.6539 be further demonstrated. The calculation of RMSD was carried out CYP21A2 −36.1355 −40.1772 −25.5294 −22.1745 −20.0655 17β-HSD1 −36.5765 −37.7614 −22.0903 −20.4887 −23.5693 using Biopolymer RMSD Calculation dialog under Discovery Studio 2019 software. Original ligand in the crystal structure of receptor protein was chosen as a reference molecule from the reference molecule 3.1. Effects of TAP and its major metabolites on key enzymes/proteins of dropdown list. TAP and its major metabolites in docking complexes of steroid hormones biosynthesis in adrenal cortex and gonads receptor protein from the list were selected as the tested molecules for the calculation of RMSD. The RMSD of the original ligand and its cor- We docked TAP and its major metabolites with the following pro- responding receptor protein was 0.0000 Å. teins: HMGR, SF-1, StAR, CYP11A1, CYP111B2, CYP117A1, CYP19A1,

N CYP21A2, and 17β-HSD1. These proteins were transcription factors, 1 RMSD = 2 transport proteins and catalytic enzymes in the biosynthesis of steroid N i i=1 (1) hormones. StAR and CYP11A1 were considered the key enzymes/proteins in steroid hormone biosynthesis. From Table 4, molecular docking

In Equation (1), δi is the distance between the ith pair of atoms. results showed that DEP and CYP11A1, CYP11B2, and CYP19A1 all had very low CDOCKER interaction energies of −39.8313, −49.5906, and −43.6539 kcal/mol, respectively. TAPO and CYP21A2 had very low 3. Results CDOCKER interaction energy of −40.1772 kcal/mol. TAP and its major metabolites had higher CDOCKER interaction energies with other en- There were several types of interactions that could be monitored zymes/proteins. Therefore, we mainly analyzed the interaction modes between a ligand and a protein in Discovery Studio 2019 (Table 3). The of TAP and its major metabolites with CYP11A1, CYP11B2, CYP19A1, categories of interactions were: Hydrogen Bonds, Electrostatic, Hydro- and CYP21A2, respectively. The results were shown in Table 5 and Figs. phobic, Miscellaneous and Unfavorable. Meanwhile, the 2D interac- S1, S2, S3, and S4, respectively. tions of the lowest binding energy poses for TAP, TAP's major meta- These results suggested that DEP, the non-specific metabolite of bolites with enzymes/proteins also could be observed in Discovery OPs, could strongly interact with the enzymes CYP11A1, CYP11B2 and Studio 2019. The 2D interactions directly reflected the interactions CYP19A1 related to steroid hormones synthesis, which might affect between TAP, TAP's major metabolites and enzymes/proteins in gra- steroid hormones (adrenocortical steroid hormones and/or sex hor- phical way, such as hydrogen bonding, Pi-Pi stacking effect, van der mones) production. TAPO could strongly interact with the enzyme Waals force, hydrophilic and hydrophobic force, which provided the- CYP21A2 related to adrenocortical steroid hormones synthesis, which oretical basis for understanding the interaction mechanism between might disturb the production of glucocorticoids and/or miner- TAP, TAP's major metabolites and enzymes/proteins. In our study, 10 alocorticoids. poses were generated for TAP and its four derivatives, TAPO, PHT, DETP and DEP, against the binding pockets of these enzymes/proteins 3.2. Effects of TAP and its major metabolites on key enzymes of and for each of the ligand. The best pose for each ligand was shown via catecholamines biosynthesis in adrenal medulla the figures and tables. We docked TAP and its major metabolites with adrenal medulla catecholamines biosynthesis enzymes TH, AAAD, DBH, and PNMT, re- Table 3 spectively. From Table 6, the results of molecular docking showed that Interaction types between TAP, TAP's major metabolites and the active sites of TAPO, DEP and AAAD had lower CDOCKER interaction energies of enzymes/proteins in this study. −38.0241 and −38.9285 kcal/mol, respectively. TAP, TAPO docked Category Sub-Category Type into PNMT have very low CDOCKER interaction energies of −42.7998 and −47.0563 kcal/mol, respectively. However, TAP and its major Hydrogen Bonds Classical Conventional Hydrogen Bond metabolites and other catecholamines biosynthetic enzymes had higher Non-Classical Carbon Hydrogen Bond Salt Bridge Salt Bridge CDOCKER interaction energy. Therefore, we mainly discussed the in- Electrostatic Charge Attractive Charges teraction of TAP and its major metabolites with AAAD and PNMT, re- Charge Salt Bridge spectively. The results were shown in Table 7, Figs. S5 and S6. Pi-Charge Pi-Cation These results indicated that TAP, TAPO, and DEP might disrupt the Pi-Charge Pi-Anion Hydrophobic Pi-Hydrophobic Pi-Pi Stacked functions and activities of the enzymes AAAD and PNMT, which were Pi-Hydrophobic Pi-Pi T-Shaped related to catecholamines biosynthesis in the adrenal medulla, thereby Pi-Hydrophobic Amide-Pi Stacked blocking its production of adrenaline. Alkyl Hydrophobic Alkyl Mixed Pi/Alkyl Hydrophobic Pi-Sigma Mixed Pi/Alkyl Hydrophobic Pi-Alkyl 3.3. Effects of TAP and its major metabolites on the enzyme iodotyrosine Miscellaneous Sulfur Pi-Sulfur deiodinase (IYD) of thyroid hormones biosynthesis in thyroid gland Sulfur Sulfur-X Lone Pair Pi-Lone Pair Unfavorable Acceptor Acceptor-Acceptor Clashes Restricted by the crystal structure of enzymes related to thyroid hormone biosynthesis and metabolism that have been resolved in the

5 F.-W. Yang, et al. Food and Chemical Toxicology 133 (2019) 110759

Table 5 Interaction sites, main types and distances between residues at the active sites of steroid hormones biosynthesis-related enzymes and TAP, TAP's major metabolites.

Receptors Ligands Interaction sites Main types Distances (Å)

CYP11A1 TAP Gln 356 Conventional hydrogen bond Lig – Gln 356 (2.48) TAPO Phe 202, Thr 354, Gln 356 Pi-Pi T shaped, Conventional hydrogen bond Lig – Phe 202 (5.98) Lig – Thr 354 (2.76) Lig – Gln 356 (2.57) PHT Val 353, Thr 354, Gln 356 Conventional hydrogen bond Lig – Val 353 (2.41) Lig – Thr 354 (2.17) Lig – Gln 356 (2.85) DETP Arg 81, Ser 352, Thr 354, Gln 356 Conventional hydrogen bond Lig – Arg 81 (2.90) Lig – Ser 352 (1.91) Lig – Thr 354 (2.82) Lig – Gln 356 (2.43) DEP Arg 81, Arg 112 Conventional hydrogen bond, Attractive charge Lig – Arg 81 (2.49) Lig – Arg 112 (5.52) CYP11B2 TAP Trp 116, Phe 130 Pi-Pi T-shaped, Pi-Pi Stacked Lig – Trp 116 (4.95) Lig – Phe 130 (4.00) TAPO Arg 110, Phe 130 Conventional hydrogen bond, Pi-Sigma, Pi-Pi Stacked Lig – Arg 110 (2.18) Lig – Phe 130 (2.80) PHT Trp 116, Phe 130, Phe 487 Pi-Pi T-shaped, Pi-Pi Stacked, Conventional hydrogen bond Lig – Trp 116 (5.21) Lig – Phe 130 (5.46) Lig – Phe 487 (2.05) DETP – – – DEP Arg 110, Arg 384, Phe 445 Conventional hydrogen bond, Salt bridge, Attractive charge, Pi-Anion Lig – Arg 110 (1.77) Lig – Arg 384 (1.79) Lig – Phe 445 (2.55) CYP19A1 TAP Arg 115 Conventional hydrogen bond Lig – Arg 115 (2.17) TAPO Arg 115, Trp 224, Cys 437 Conventional hydrogen bond, Pi-Pi Stacked, Lig – Arg 115 (2.39) Lig – Trp 224 (5.40) Lig – Cys 437 (3.00) PHT Arg 115, Met 374 Conventional hydrogen bond, Attractive charge Lig – Arg 115 (2.66) Lig – Met 374 (2.00) DETP – – – DEP Arg 115, Arg 145, Arg 435, Ala 438 Attractive charge, Salt bridge, Conventional hydrogen bond Lig – Arg 115 (2.56) Lig – Arg 145 (2.67) Lig – Arg 435 (4.94) Lig – Ala 438 (2.03) CYP21A2 TAP Trp 202 Pi-Sulfur Lig – Trp 202 (4.46) TAPO Trp 202 Pi-Pi Stacked Lig – Trp 202 (4.24) PHT Asp 107, Asp 288 Conventional hydrogen bond Lig – Asp 107 (2.00) Lig – Asp 288 (2.13) DETP – – – DEP Trp 202 Pi-Anion Lig – Trp 202 (4.91)

Table 6 docked into HSA could form very low CDOCKER interaction energies of The lowest interaction energy (kcal/mol) between TAP, TAP's major metabo- −39.9807, −44.1985 and −40.1406 kcal/mol, respectively. TAPO lites and catecholamines biosynthesis-related enzymes. formed a very low CDOCKER interaction energy of −40.2857 kcal/mol Receptors TAP TAPO PHT DETP DEP with SHBG. DEP formed very low CDOCKER interaction energy of −40.5303 kcal/mol with TBG. Therefore, we mainly listed the inter- TH −35.2452 −38.8217 −25.9069 −24.6320 −24.9642 action modes of TAP and its major metabolites with HSA, SHBG, and AAAD −31.6843 −38.0241 −21.5726 −24.0328 −38.9285 TBG, respectively. These results were shown in Table 11 and Figures S8- DBH −22.0572 −27.4095 −14.3949 −16.2235 −28.5891 PNMT −42.7998 −47.0563 −26.8873 −27.9821 −19.9956 S10 respectively. Based on these results, we could speculate that TAP, TAPO, and DEP could affect the binding transport of HSA with adrenocortical hor- PDB database, we could only dock TAP and its main metabolites with mones, sex hormones, and thyroid hormones through intermolecular the thyroid hormones biosynthesis enzyme iodotyrosine deiodinase interaction with HSA. Meanwhile, TAPO could disturb the circulation of (IYD) in the thyroid gland. Table 8 showed that DEP and IYD had very sex hormones by binding with SHBG, and DEP might affect the trans- low CDOCKER interaction energy of −45.7062 kcal/mol. The docking port of thyroid hormones in the blood by interacting with TBG. complexes of TAP, TAPO, PHT, DETP, DEP and IYD and the corresponding IYD interacting amino acid residues were shown in Table 9 3.5. Effects of TAP and its major metabolites on hormone receptors and Fig. S7. These demonstrated the interaction of TAP and its major metabolites with amino acids of IYD protein, respectively. DEP could We docked TAP and its major metabolites with hormone receptors, interact strongly with IYD, indicating that DEP might be a potential such as GR, MR, AR, CAR, ERα, ERβ, ERRα, ERRγ, PR, TRα, TRβ, thyroid hormone endocrine disruptor. tβ1AR, β2AR, D2DR, D3DR, and D4DR, respectively. The molecular docking results in Table 12 showed that TAP and GR, AR, TRα, and TRβ 3.4. Effects of TAP and its major metabolites on hormone transport proteins could form very low CDOCKER interaction energies of −36.8425, −37.9152, −40.2179, and −41.7089 kcal/mol, respectively. TAPO We docked TAP and its major metabolites with hormone transport formed very low CDOCKER interaction energies with more hormone binding proteins in blood such as HSA, CBG, SHBG, TBG, and TTR, receptors. For example, TAPO formed low interaction energies of respectively. The results of Table 10 showed that TAP, TAPO, DEP −38.0438, −38.6282, −40.0815, −39.2319, −38.6160, −40.2356,

6 F.-W. Yang, et al. Food and Chemical Toxicology 133 (2019) 110759

Table 7 Interaction sites, main types and distances between residues at the active sites of catecholamine hormones biosynthesis-related enzymes and TAP, TAP's major metabolites.

Receptors Ligands Interaction sites Main types Distances (Å)

AAAD TAP Tyr 79, Lys 303, Arg 447 Pi-Pi T-shaped, Conventional hydrogen bond, Pi-Cation Lig – Tyr 79 (5.64) Lig – Lys 303 (1.81) Lig – Arg 447 (3.17) TAPO Tyr 79, Lys 303, Arg 447 Pi-Pi T-shaped, Conventional hydrogen bond, Pi-Cation Lig – Tyr 79 (5.46) Lig – Lys 303 (1.71) Lig – Arg 447 (3.20) PHT Lys 303 Pi-Cation Lig – Lys 303 (3.09) DETP Lys 303 Conventional hydrogen bond Lig – Lys 303 (1.89) DEP Thr 246, Lys 303 Conventional hydrogen bond, Attractive charge Lig – Thr 246 (2.39) Lig – Lys 303 (2.35) PNMT TAP Tyr 27, Gly 79, Asp 101, Phe 102, Asn 106 Pi-Pi Stacked, Unfavorable Acceptor-Acceptor, Pi-Anion, Pi-Pi T-shaped, Lig – Tyr 27 (5.80) Conventional hydrogen bond Lig – Gly 79 (2.99) Lig – Asp 101 (3.54) Lig – Phe 102 (4.70) Lig – Asn 106 (2.59) TAPO Tyr 27, Phe 30, Tyr 35, Asp 101, Phe 102, Cys Pi-Pi Stacked, Pi-Pi T-shaped, Conventional hydrogen bond, Pi-Anion, Pi-Sulfur Lig – Tyr 27 (5.21) 183 Lig – Phe 30 (5.57) Lig – Tyr 35 (2.70) Lig – Asp 101 (3.42) Lig – Phe 102 (4.64) Lig – Cys 183 (5.30) PHT Tyr 27, Tyr 35, Ser 80, Thr 83 Pi-Pi T-shaped, Conventional hydrogen bond, Pi-Pi Stacked Lig – Tyr 27 (5.18) Lig – Tyr 35 (2.33) Lig – Ser 80 (2.04) Lig – Thr 83 (2.30) DETP Tyr 35, Phe 182 Conventional hydrogen bond Lig – Tyr 35 (2.71) Lig – Phe 182 (1.95) DEP Tyr 35 Conventional hydrogen bond Lig – Tyr 35 (2.81)

Table 8 Table 10 The lowest interaction energy (kcal/mol) between TAP, TAP's major metabo- The lowest interaction energy (kcal/mol) between TAP, TAP's major metabolites and the thyroid hormones biosynthesis-related enzyme IYD. lites and hormone transport proteins.

Receptor TAP TAPO PHT DETP DEP Receptors TAP TAPO PHT DETP DEP

IYD −21.4739 −31.4681 −15.7375 −16.2651 −45.7062 HSA −39.9807 −44.1985 −25.3253 −23.7186 −40.1406 CBG −30.7416 −33.2241 −22.0712 −19.3301 −18.2183 SHBG −37.1562 −40.2857 −25.6923 −24.2684 −21.2266 −40.5868, −42.7165, −41.6788 kcal/mol with GR, MR, AR, CAR, TBG −34.1400 −35.8789 −22.3594 −20.5038 −40.5303 TTR −35.2280 −37.1439 −20.6632 −23.8367 −30.1745 ERα, PR, TRα, TRβ, and β1AR, respectively. The interaction energies of PHT and DETP with these hormone receptors were higher. DEP formed a lower CDOCKER interaction energy of −37.7211 kcal/mol with TRα. sapiens) and turkeys (Meleagris gallopavo) was approximately 54.93%. Therefore, we mainly described the interaction patterns of TAP and its The sequence alignment results showed that the amino acids that in- major metabolites with GR, MR, AR, CAR, ERα, PR, TRα, TRβ, and teracted with TAP and its major metabolites, in these two proteins were β1AR, respectively. These results were shown in Table 13 and Figs. S11, consistent and conservative (Fig. S20). S12, S13, S14, S15, S16, S17, S18, and S19, respectively. In addition, Overall, TAP might disrupt these three hormone receptors: GR, TRα the homology of β1AR amino acid sequences between humans (Homo and TRβ. TAPO might interfere with the normal binding of these

Table 9 Interaction sites, main types and distances between residues at the active sites of thyroid hormones biosynthesis-related enzyme IYD and TAP, TAP's major metabolites.

Receptor Ligands Interaction sites Main types Distances (Å)

IYD TAP Arg 100, Arg 104 Conventional hydrogen bond, Pi-Anion Lig – Arg 100 (2.58) Lig – Arg 104 (3.24) TAPO Arg 100, Arg 104 Conventional hydrogen bond Lig – Arg 100 (1.79) Lig – Arg 104 (2.59) PHT Arg 100, Ser 102, Arg 104, Thr 237 Conventional hydrogen bond, Pi-Anion Lig – Arg 100 (2.84) Lig – Ser 102 (2.65) Lig – Arg 104 (3.59) Lig – Thr 237 (1.88) DETP Arg 104, Lys 182, Tyr 184 Conventional hydrogen bond Lig – Arg 104 (1.98) Lig – Lys 182 (2.43) Lig – Tyr 184 (2.90) DEP Arg 100, Arg 101, Ser 102, Arg 279 Conventional hydrogen bond, Attractive charge, Salt bridge Lig – Arg 100 (1.88) Lig – Arg 101 (4.46) Lig – Ser 102 (2.07) Lig – Arg 279 (2.60)

7 F.-W. Yang, et al. Food and Chemical Toxicology 133 (2019) 110759

Table 11 Interaction sites, main types and distances between residues at the active sites of hormone transport proteins and TAP, TAP's major metabolites.

Receptors Ligands Interaction sites Main types Distances (Å)

HSA TAP Lys 199, Arg 222 Conventional hydrogen bond Lig – Lys 199 (2.66) Lig – Arg 222 (3.00) TAPO Lys 199 Conventional hydrogen bond Lig – Lys 199 (2.05) PHT Arg 257, Ser 287, Ile 290 Attractive charge, Conventional hydrogen bond, Amide-Pi Stacked Lig – Arg 257 (4.58) Lig – Ser 287 (1.87) Lig – Ile 290 (4.54) DETP Arg 257 Conventional hydrogen bond Lig – Arg 257 (2.12) DEP Lys 199, Arg 218, Arg 222 Conventional hydrogen bond, Attractive charge Lig – Lys 199 (1.85) Lig – Arg 218 (2.82) Lig – Arg 222 (2.61) SHBG TAP Phe 67, Asn 82, Met 139 Pi-Pi Stacked, Conventional hydrogen bond, Pi-Sulfur Lig – Phe 67 (4.89) Lig – Asn 82 (2.59) Lig – Met 139 (4.16) TAPO Phe 67, Asn 82, Met 139 Pi-Pi Stacked, Conventional hydrogen bond, Pi-Sulfur Lig – Phe 67 (4.84) Lig – Asn 82 (2.17) Lig – Met 139 (4.29) PHT Asp 65, Phe 67, Asn 82 Pi-Pi Stacked, Conventional hydrogen bond Lig – Asp 65 (2.15) Lig – Phe 67 (4.08) Lig – Asn 82 (2.78) DETP – – – DEP Phe 67 Pi-Anion Lig – Phe 67 (4.78) TBG TAP Tyr 20, Lys 270, Asn 273, Arg 381 Pi-Cation, Conventional hydrogen bond Lig – Tyr 20 (4.67) Lig – Lys 270 (3.17) Lig – Asn 273 (3.04) Lig – Arg 381 (3.44) TAPO Arg 381 Pi-Sigma Lig – Arg 381 (2.75) PHT Gln 238 Conventional hydrogen bond Lig – Gln 238 (1.98) DETP Asn 273 Conventional hydrogen bond Lig – Asn 273 (1.99) DEP Ser 266, Lys 270 Conventional hydrogen bond, Attractive charge Lig – Ser 266 (2.55) Lig – Lys 270 (2.75)

Table 12 4. Discussion The lowest interaction energy (kcal/mol) between TAP, TAP's major metabolites and hormone receptors. 4.1. Overview of TAP and its major degradation metabolites Receptors TAP TAPO PHT DETP DEP Organophosphate pesticides, which were widely used in agriculture, GR −36.8425 −38.0438 −21.8705 −20.8930 −21.3411 were highly toxic and commonly ingested with suicidal intent in de- MR −35.5446 −38.6282 −22.7501 −21.5424 −25.8509 veloping countries (Henretig et al., 2019). Similarly, TAP has been AR −37.9152 −40.0815 −23.0466 −22.0322 −24.1372 CAR −36.3142 −39.2319 −20.4643 −20.8157 −20.8015 widely used globally for more than 40 years (Holden et al., 2001; JMPR, ERα −34.9721 −38.6160 −24.3489 −21.2301 −22.1135 2002). China's pesticide risk monitoring results also indicated that TAP ERβ −33.3428 −34.8708 −26.2235 −23.3019 −26.1649 had high risks or risk uncertainty in terms of toxicity, residue, and ERRα −31.3311 −34.1177 −25.3990 −23.7727 −22.8410 environmental safety. The Ministry of Agriculture of China has banned ERRγ −25.5320 −27.9839 −27.5525 −24.5199 −26.6702 the use of TAP in vegetables and started re-evaluation study of TAP. PR −36.6287 −40.2356 −24.5775 −21.9644 −26.5485 TRα −40.2179 −40.5868 −30.3521 −22.5194 −37.7211 However, TAP was still registered in China for pest control in rice, TRβ −41.7089 −42.7165 −24.8326 −22.2293 −30.8696 cotton, and grassland. And TAP was often detected in agricultural β1AR −35.2497 −41.6788 −26.0393 −23.9079 −22.3257 products, foods, and environmental soil and water samples (Bhandari β2AR - 35.5131 −36.7502 −24.5873 −22.1916 −24.1935 et al., 2019; Hong et al., 2019; Kumari and John, 2019). The biological D2DR −35.6015 −37.3540 −21.2402 −22.1884 −21.1554 D3DR −33.9438 −37.0953 −25.2388 −23.5515 −24.8556 half-life of TAP in the body was short (JMPR, 2002), and TAP was ra- D4DR −37.7654 −36.5579 −21.6463 −21.5246 −18.5749 pidly metabolized to the specific metabolites TAPO and PHT, and the non-specific DAP metabolites DETP and DEP under the action of aseries of metabolic detoxification enzymes (Bock and Their, 1976; Schwalbe- receptors GR, MR, AR, CAR, ERα, PR, TRα, TRβ, and β1AR to their Fehl and Schmidt, 1986; Wang et al., 2015). respective native ligands. Although the binding of PHT and PR had Approximately 75% of OPs registered in the market were metabo- higher binding interaction energy, there were many strong chemical lized to the DAP metabolites (Shomar et al., 2014; Ueyama et al., 2015). bonds between them. Thus, PHT might have adverse effects on the At the same time, OPs were also degraded into DAPs such as DETP and normal physiological function of PR. DEP might interact with TRα and DEP due to the metabolism of the plants and the environmental factors should be able to be considered as a potential thyroid hormone dis- and were present in fruits and vegetables (Zhang et al., 2008). A certain ruptor. concentration of chlorpyrifos pesticide was sprayed on the green string We also verified the accuracy of the molecular docking results to bean grown in the field, and the residues of DEP could still be detected ensure the accuracy of the parameters and method selection during the in the samples collected on the 21st day after spraying (Pan et al., molecular docking process. The evaluation index was the value of 2012). Interestingly, some organophosphorus flame retardants and RMSD. The smaller the RMSD was, the closer the docking results of TAP plasticizers could also be degraded and metabolized to DEP (Chu and and its major metabolites with the target proteins were to the structure Letcher, 2018; Li et al., 2017; Reemtsma et al., 2011; Sun et al., 2018). of the original ligands and the target proteins. See Table S1 for the The correlation between OPs with diseases established by epidemiolo- specific results. As shown in Table S1, the CDOCKER molecular docking gical studies mainly based on the contents of their metabolites in- results of Discovery Studio 2019were credible and acceptable. cluding DEP in the urine (Katsikantami et al., 2019). Epidemiologic

8 F.-W. Yang, et al. Food and Chemical Toxicology 133 (2019) 110759

Table 13 Interaction sites, main types and distances between residues at the active sites of hormone receptors and TAP, TAP's major metabolites.

Receptors Ligands Interaction sites Main types Distances (Å)

GR TAP Asn 564, Phe 623, Tyr 735, Cys 736 Conventional hydrogen bond, Pi-Pi T-shaped, Pi-Sulfur Lig – Asn 564 (2.68) Lig – Phe 623 (5.01) Lig – Tyr 735 (5.33) Lig – Cys 736 (2.08) TAPO Leu 563, Tyr 735, Cys 736 Pi-Pi T-shaped, Amide-Pi stacked, Pi-Sulfur Lig – Leu 563 (4.73) Lig – Tyr 735 (5.93) Lig – Cys 736 (3.62) PHT Met 560, Asn 564, Phe 623, Met 646 Conventional hydrogen bond, Pi-Pi T-shaped, Pi-Sulfur Lig – Met 560 (2.25) Lig – Asn 564 (2.07) Lig – Phe 623 (5.23) Lig – Met 646 (5.19) DETP Asn 564 Conventional hydrogen bond Lig – Asn 564 (1.93) DEP – – – MR TAP Phe 829 Pi-Pi T-shaped bond Lig – Phe 829 (5.01) TAPO Met 807, Phe 829 Pi-Sulfur, Pi-Pi T-shaped Lig – Met 807 (4.00) Lig – Phe 829 (5.04) PHT Asn 770, Cys 942 Conventional hydrogen bond, Pi-Lone pair, Pi-Sulfur Lig – Asn 770 (2.26) Lig – Cys 942 (4.57) DETP Asn 770 Conventional hydrogen bond Lig – Asn 770 (1.91) DEP Arg 817 Attractive charge Lig – Arg 817 (4.75) AR TAP Leu 704, Phe 764, Met 780 Pi-Sigma, Pi-Sulfur Lig – Leu 704 (2.70) Lig – Phe 764 (4.59) Lig – Met 780 (4.94) TAPO – – – PHT Asn 705, Phe 764 Conventional hydrogen bond, Pi-Pi T-shaped Lig – Asn 705 (1.87) Lig – Phe 764 (5.98) DETP Asn 705 Conventional hydrogen bond Lig – Asn 705 (2.13) DEP – – – CAR TAP Phe 161, His 203, Tyr 326 Pi-Pi T-shaped, Pi-Sulfur, Pi-Pi Stacked Lig – Phe 161 (5.03) Lig – His 203 (4.92) Lig – Tyr 326 (4.98) TAPO Phe 161, His 203, Tyr 326 Pi-Pi T-shaped, Pi-Pi Stacked Lig – Phe 161 (5.10) Lig – His 203 (4.70) Lig – Tyr 326 (4.98) PHT Phe 161, Thr 225, Phe 243 Pi-Pi T-shaped, Conventional hydrogen bond, Pi-Pi T-shaped Lig – Phe 161 (4.88) Lig – Thr 225 (2.04) Lig – Phe 243 (5.69) DETP Tyr 224, Thr 225 Pi-Sulfur, Conventional hydrogen bond Lig – Tyr 224 (5.86) Lig – Thr 225 (1.90) DEP Phe 234 Pi-Anion Lig – Phe 234 (3.87) ERα TAP Met 343, Thr 347, Met 528 Conventional hydrogen bond, Pi-Sulfur, Pi-Sulfur Lig – Met 343 (5.42) Lig – Thr 347 (2.72) Lig – Met 528 (4.88) TAPO Met 528 Pi-Sulfur Lig – Met 528 (5.38) PHT Arg 394, Phe 404 Conventional hydrogen bond, Attractive charge, Pi-Pi T-shaped Lig – Arg 394 (2.22) Lig – Phe 404 (5.09) DETP Thr 347, Cys 530 Conventional hydrogen bond Lig – Thr 347 (2.62) Lig – Cys 530 (2.45) DEP – – – PR TAP Met 756 Sulfur-X Lig – Met 756 (3.26) TAPO Met 759, Phe 778, Met 909 Pi-Sigma, Pi-Pi T-shaped, Pi-Sulfur Lig – Met 759 (2.80) Lig – Phe 778 (5.36) Lig – Met 909 (5.44) PHT Gln 725, Met 756, Met 759, Arg 766, Phe 778, Met 801 Conventional hydrogen bond, Pi-Sulfur, Attractive charge, Pi-Pi T-shaped Lig – Gln 725 (2.17) Lig – Met 756 (4.49) Lig – Met 759 (4.37) Lig – Arg 766 (2.36) Lig – Phe 778 (5.38) Lig – Met 801 (5.40) DETP Leu 718, Trp 755 Conventional hydrogen bond, Pi-Sulfur Lig – Leu 718 (1.97) Lig – Trp 755 (5.61) DEP Gln 725 Conventional hydrogen bond Lig – Gln 725 (2.44) TRα TAP Met 256, Met 388 Pi-Sulfur, Sulfur-X Lig – Met 256 (3.03) Lig – Met 388 (5.72) TAPO Phe 218 Unfavorable acceptor-acceptor Lig – Phe 218 (2.80) PHT Arg 228, Ser 277 Conventional hydrogen bond, Attractive charge Lig – Arg 228 (1.93) Lig – Ser 277 (2.33) DETP – – – DEP Arg 228, Ser 277 Conventional hydrogen bond, Attractive charge Lig – Arg 228 (2.48) Lig – Ser 277 (2.16) (continued on next page)

9 F.-W. Yang, et al. Food and Chemical Toxicology 133 (2019) 110759

Table 13 (continued)

Receptors Ligands Interaction sites Main types Distances (Å)

TRβ TAP Met 313, Met 442 Pi-Sulfur Lig – Met 313 (5.82) Lig – Met 442 (4.83) TAPO Met 313, Met 442 Pi-Sulfur Lig – Met 313 (5.73) Lig – Met 442 (5.21) PHT Met 313, Asn 331 Conventional hydrogen bond Lig – Met 313 (1.85) Lig – Asn 331 (2.35) DETP Met 310 Sulfur-X Lig – Met 310 (3.50) DEP Arg 282, Asn 331 Attractive charge, Conventional hydrogen bond Lig – Arg 282 (5.14) Lig – Asn 331 (2.00) β1AR TAP Trp 117, Phe 201, Phe 306 Pi-Sulfur, Conventional hydrogen bond, Pi-Pi T-shaped Lig – Trp 117 (4.74) Lig – Phe 201 (2.64) Lig – Phe 306 (5.05) TAPO Phe 201, Ser 212, Phe 306 Pi-Pi T-shaped bond, Conventional hydrogen bond Lig – Phe 201 (4.93) Lig – Ser 212 (2.30) Lig – Phe 306 (5.10) PHT Asp 121, Phe 307, Asn 329 Conventional hydrogen bond, Pi-Pi T-shaped bond Lig – Asp 121 (1.94) Lig – Phe 307 (5.59) Lig – Asn 329 (2.30) DETP Asp 121 Conventional hydrogen bond Lig – Asp 121 (2.05) DEP Asn 310 Conventional hydrogen bond Lig – Asn 310 (2.97) studies established the association between OPs and disturbed hormone and Beg, 2017), SHBG (Hazarika et al., 2019; Liu et al., 2016; Sheikh levels based on urinary DEP measurements, especially the disorder of and Beg, 2019; Sheikh et al., 2016b, 2016c), TBG (Ren et al., 2016), and serum sex hormone levels and thyroid hormone levels in OPs exposed TTR (Grimm et al., 2013; Hill et al., 2018; Kar et al., 2017; Ren et al., population (Bernieri et al., 2019; Omoike et al., 2015; Panuwet et al., 2016; Xin et al., 2018; Zhang et al., 2015, 2016a; Zhang et al., 2018; Shrestha et al., 2018; Wang et al., 2017a,b). 2018a,b,c) in silico, affecting hormone binding and transport, and the levels of bioavailable free hormones. 4.2. Endocrine disrupting effects of TAP in vivo and in vitro Our results also suggested that DEP, the non-specific metabolite of organophosphorus pesticides, can strongly interact with the enzymes TAP exposure can affect the thyroid hormone levels and theex- CYP11A1, CYP11B2 and CYP19A1 related to steroid hormones synth- pression of TRα, TRβ, Dio1, Dio2, tsh, ERα, ERβ1, ERβ2 in zebrafish esis, which might affect steroid hormones (adrenocortical steroid hor- embryos (Wu et al., 2018). In vitro cell experiments showed that TAP mones and/or sex hormones) production. TAPO can strongly interact had the potentials to disrupt the estrogen receptor (ER), aromatic hy- with the enzyme CYP21A2 related to adrenocortical steroid hormones drocarbon receptor, constitutive androstane receptor (CAR), pregnane synthesis, which might disturb the production of glucocorticoids and/or X receptor, and vitamin D receptor signaling pathways (https:// mineralocorticoids. Previous studies have also found that CYP11A1, pubchem.ncbi.nlm.nih.gov/compound/32184)(Table S2). CYP11B2, CYP19, and CYP21 were targets of EDCs-induced adreno- The effects of TAP on reproductive activity were mainly explored in cortical toxicity and endocrine disruption in steroidogenic pathway female rats (Sharma et al., 2015a) and the effects on the offsprings were (Harvey et al., 2007; Harvey, 2016). DEP can interact strongly with also studied under pre-conceptional exposure to TAP (Sharma et al., IYD, indicating that DEP might be a potential thyroid hormone endo- 2015b; Bhanot and Sangha, 2018). TAP exposure altered serum content crine disruptor. In addition, our study indicated that TAP, TAPO and of estrogen and progesterone, elevated the peroxidation and apoptosis DEP might disrupt the functions and activities of the enzymes AAAD in ovary at the dose of 1/10th, 1/20th, and 1/40th of LD50 (Sharma and PNMT, which were related to catecholamines biosynthesis in et al., 2015a). In utero and lactational exposure to acceptable daily in- adrenal medulla (Kvetnansky et al., 2009, 2013), thereby disturbing the take level of TAP influenced testis development and functions in the production of adrenaline in the adrenal medulla. The neonicotinoid male offsprings, which were characterized by a significant fall insperm insecticide imidacloprid can facilitate TH transcription and PNMT count, sperm motility, plasma testosterone levels, and histopathological mRNA expression to enhance catecholamine synthesis in PC12D cells alterations in testis (Bhanot and Sangha, 2018). However, non-sig- (Kawahata and Yamakuni, 2018). High dietary phosphoric acid in- nificant changes in reproductive indices such as gestational length and creased plasma phosphate concentration, blood pressure, and pulse pup viability were observed in the offsprings in female rats under count. This mechanism might involve changes in sympathetic adrena- preconceptional exposure of 30 days to 1/10th and 1/20th of LD50 of line activity (Mohammad et al., 2018). Phosphoric acid and DEP were TAP (Sharma et al., 2015b). similar in chemical structure. Therefore, TAP and its major metabolites might exert endocrine disrupting effects by disturbing the biosynthesis processes of hormones in the endocrine glands. 4.3. Potential disrupting effects of TAP and its major metabolites on Based on these results of our research, we speculate that TAP, TAPO, endocrine hormones biosynthesis and transport and DEP can affect the binding transport of HSA with adrenocortical hormones, sex hormones, and thyroid hormones through inter- EDCs, also known as endocrine disruptors, were chemicals that molecular interaction with HSA. TAPO can also disturb the circulation could alter endocrine function by mimicking, blocking, or interfering of sex hormones by binding with SHBG, and DEP might affect the with the production, metabolism, or action of hormones in the body transport of thyroid hormones in the blood by interacting with TBG. (The Lancet Diabetes & Endocrinology, 2019). Molecular docking can Predictably, TAP and its major metabolites might also interact with reveal the potential of EDCs (phthalate esters) to inhibit the enzymes of hormone-binding globulins and transport proteins in the blood, thereby the glucocorticoid biosynthesis pathway (Ahmad et al., 2017), and affecting the levels of free hormones available in the peripheral circu- unraveling the molecular targets of bisphenol A and S in the thyroid lation. Similarly, it can interfere with the negative feedback regulation gland (Berto-Júnior et al., 2018). EDCs also disturbed hormone-binding of endocrine hormones. globulins and transport proteins in the blood, such as the interfering effects of EDC on HSA(Hill et al., 2018; Peng et al., 2016), CBG (Sheikh

10 F.-W. Yang, et al. Food and Chemical Toxicology 133 (2019) 110759

4.4. Potential disrupting effects of TAP and its major metabolites onthe in silico. All three compounds (TAP, TAPO, and DEP) showed high binding of endocrine hormone receptor proteins binding affinity with more proteins and enzymes than PHT andDETP. TAP might interfere with the endocrine function of the adrenal glands, In addition to being regulated by their natural hormone ligands including the biosynthesis of corticosteroids and catecholamines. TAP binding, nuclear hormone receptors were also affected and regulated by might also bind strongly with GR and TR, affecting the normal phy- exogenous environmental contaminants (Wang et al., 2017a,b), EDCs siological function of these hormone receptors. TAPO might disrupt the (Balaguer et al., 2017; Le Maire et al., 2010; Ruiz et al., 2017; Sharma normal binding of AR, ER, PR, and β1AR to their natural hormone li- et al., 2018; Usman and Ahmad, 2019), foods and their components gands. The binding of PHT and endocrine hormone-related proteins and (Cozzini and Spyrakis, 2017; Goto, 2019; Zhang et al., 2018c), drugs enzymes had higher binding interaction energies, PHT and PR could (Cozzini and Spyrakis, 2017), and toxics (Choudhuri et al., 2017), form many strong chemical bonds. Analysis of the molecular docking among others, and can cause transcription and expression of the results of DETP showed that there was no strong molecular interaction downstream target genes, leading to a series of physiological and pa- between DETP and all endocrine hormone-related proteins and en- thological phenotypes. Steroid hormone receptors (GR, MR, AR, CAR, zymes involved in this study, suggesting that DETP was unlikely to be ERα, ERβ, and PR) and thyroid hormone receptors (TRα and TRβ) also an EDC. DEP might affect biosynthesis of steroid hormones (adreno- played a series of important roles in maintaining normal growth and cortical steroid hormones and/or sex hormones) and thyroid hormones. development, reproduction, energy metabolism, inflammation, and At the same time, DEP might disrupt the binding and transport of immunity in humans and animals (Conroy-Ben et al., 2018; Lu et al., thyroid hormones in the blood and the normal binding of thyroid 2018; Pande et al., 2019; Weikum et al., 2017; Zhang et al., 2018b). hormones to their receptors. Together, these suggested that TAP and EDCs, including pesticides, could interfere with hormone receptors in DEP might have endocrine disrupting activities and were potential vivo, in vitro and in silico (Chen et al., 2018), such as GR (Khan et al., EDCs. 2017; Prasanth et al., 2010; Sarath Josh et al., 2016; Zhang et al., TAP remains a commonly used organophosphorus pesticide in many 2016c), MR (Khan et al., 2017), AR (Chen et al., 2019; Conroy-Ben developing countries, with high residual and high exposure risks. DEP et al., 2018; Khan et al., 2017; Sarath Josh et al., 2016), CAR (Verma was a non-specific metabolite of diethyl thiophosphate and diethyl et al., 2017), ERα (Conroy-Ben et al., 2018; Zhang et al., 2018b), PR phosphate organophosphorus pesticides, and even some organopho- (Khan et al., 2017; Sarath Josh et al., 2016; Sheikh et al., 2016a), and sphorus flame retardants and plasticizers could also be metabolized to TR (Li et al., 2010; Lu et al., 2018; Ren et al., 2015; Xin et al., 2018; DEP. Moreover, DEP not only can be produced by metabolism in the Zhang et al., 2016b), among others, thus affecting their biological ef- human body, but can be present in agricultural products, foods, and the fects. Dysfunction of the monoaminergic neurotransmission was im- environment. The human body could be exposed to DEP through var- plicated in major depressive disorder and other neuropsychiatric con- ious ways such as diet and the environment. Therefore, based on our ditions (Liu et al., 2018). Therefore, the study of the interaction findings, it was necessary to be more cautious and rigorous in theusage between EDCs and hormone receptors was an important technique for and residue limit standards of organophosphorus chemicals. screening EDCs. The molecular docking simulation method, as a computational toxicology research approach based on the interaction be- Conflicts of interest tween EDCs and receptors, has been increasingly widely applied (Cavaliere and Cozzini, 2018; Chen et al., 2018; Vuorinen et al., 2015). The authors declare that no conflicts of interest in this article. By investigating the binding abilities of TAP and its major metabolites to hormone receptors, we found that TAP might disrupt these Declaration of interests three hormone receptors: GR, TRα and TRβ. TAPO might interfere with the normal binding of these receptors GR, MR, AR, CAR, ERα, PR, TRα, The authors declare that they have no known competing financial TRβ, and β1AR to their respective native ligands. Although the binding interests or personal relationships that could have appeared to influ- of PHT and PR had higher binding interaction energy, there were many ence the work reported in this paper. strong chemical bonds between them, so PHT might have adverse effects on the normal physiological function of PR. DEP might interact Acknowledgements with TRα and act as a potential thyroid hormone disruptor. Obviously, TAP and its major metabolites might also affect the transcription and This work was financially supported by the National Key Research expression of downstream genes by destroying the binding abilities of and Development Program of China (grant number 2017YFC1601803) hormone receptors to their native hormone ligands, leading to a series and Beijing Municipal Science and Technology Commission project of adverse biological effects. Further, we could also give an example of (grant number D171100008017003). And authors deeply thank Dr. validation here. When docking RU486, an antagonist of GR (Chen et al., Zhan-Hui Wang and Dr. Ming-Gang Liu (Beijing Advanced Innovation 2018; Weikum et al., 2017), with GR (Fig. S21), it was found that Center for Food Nutrition and Human Health, College of Veterinary RU486 could form the Pi-Pi T-shaped, Pi-Sulfur with Phe623 and Medicine, China Agricultural University) for their generous support of Cys736 of GR, respectively. RU486 also formed Alkyl, Pi-Alkyl bonds the molecular docking tools of Discovery Studio. with GR at the sites including Leu563 and Tyr735 in GR. These sites were in common with TAP, indicating that TAP might inhibit the Appendix A. Supplementary data binding of glucocorticoids with GR. Supplementary data to this article can be found online at https:// 5. Conclusions doi.org/10.1016/j.fct.2019.110759.

The endocrine disrupting effects of TAP led to only a few research References advances in vivo and in vitro. The correlation between DEP levels in human urine and endocrine hormone disorders had also been in- Conroy-Ben, O., Garcia, I., Teske, S.S., 2018. In silico binding of 4, 4'‐bisphenols predicts in vestigated in several epidemiological studies, but there was no direct vitro estrogenic and antiandrogenic activity. Environ. Toxicol. 33, 569–578. https:// doi.org/10.1002/tox.22539. causal supporting evidence. The purpose of this study was to under- The Lancet Diabetes & Endocrinology, 2019. EDCs: regulation still lagging behind evi- stand the mechanism of molecular interactions of TAP and its four dence. The Lancet Diabetes Endo 7, 325. https://doi.org/10.1016/S2213-8587(19) major metabolites (TAPO, PHT, DETP, and DEP) with endocrine hor- 30114-7. Adedara, I.A., Owoeye, O., Ajayi, B.O., Awogbindin, I.O., Rocha, J.B., Farombi, E.O., mone-related proteins and enzymes using molecular docking simulation

11 F.-W. Yang, et al. Food and Chemical Toxicology 133 (2019) 110759

2018. Diphenyl diselenide abrogates chlorpyrifos-induced hypothalamic-pituitary- Choudhuri, S., Patton, G.W., Chanderbhan, R.F., Mattia, A., Klaassen, C.D., 2017. From testicular axis impairment in rats. Biochem. Bioph. Res. Co. 503, 171–176. https:// Classical Toxicology to Tox21: some critical conceptual and technological advances doi.org/10.1016/j.bbrc.2018.05.205. in the molecular understanding of the toxic response beginning from the last quarter Ahmad, S., Khan, M.F., Parvez, S., Akhtar, M., Raisuddin, S., 2017. Molecular docking of the 20th century. Toxicol. Sci. 161, 5–22. https://doi.org/10.1093/toxsci/kfx186. reveals the potential of phthalate esters to inhibit the enzymes of the glucocorticoid Chu, S., Letcher, R.J., 2018. A mixed-mode chromatographic separation method for the biosynthesis pathway. J. Appl. Toxicol. 37, 265–277. https://doi.org/10.1002/jat. analysis of dialkyl phosphates. J. Chromatogr. A 1535, 63–71. https://doi.org/10. 3355. 1016/j.chroma.2017.12.069. Ahmad, S.N., Nasir, M., Naureen, A., Andleeb, S., Raees, K., Abbas, T., Younis, A., Ahmad, Cozzini, P., Spyrakis, F., 2017. Nuclear receptors: from drugs to food, and from in silico to K.R., 2018. Histopathological and micrometric studies of diazinon exposure on in vitro. Nucl. Recept. Res. 4, 101319. https://doi.org/10.11131/2017/101319. thyroid and parathyroid tissues in mice. Biologia (Pakistan) 64, 27–36. da Silva Hage-Melim, L.I., Poiani, J.G.C., Boylan, F., 2019. In silico study of the me- Akande, M.G., Shittu, M., Uchendu, C., Yaqub, L.S., 2016. Taurine ameliorated thyroid chanism of action, pharmacokinetic and toxicological properties of some N-methy- function in rats co-administered with chlorpyrifos and lead. Vet. Res. Commun. 40, lanthranilates and their analogs. Food Chem. Toxicol. 131, 110556. https://doi.org/ 123–129. https://doi.org/10.1007/s11259-016-9662-9. 10.1016/j.fct.2019.06.003. Balaguer, P., Delfosse, V., Grimaldi, M., Bourguet, W., 2017. Structural and functional Duan, Y., Guan, N., Li, P., Li, J., Luo, J., 2016. Monitoring and dietary exposure assess- evidences for the interactions between nuclear hormone receptors and endocrine ment of pesticide residues in cowpea (Vigna unguiculata L. Walp) in Hainan, China. disruptors at low doses. Comptes Rendus Biol. 340, 414–420. https://doi.org/10. Food Control 59, 250–255. https://doi.org/10.1016/j.foodcont.2015.05.036. 1016/j.crvi.2017.08.002. Erickson, J.A., Jalaie, M., Robertson, D.H., Lewis, R.A., Vieth, M., 2004. Lessons in mo- Beaulieu, J.-M., Gainetdinov, R.R., 2011. The physiology, signaling, and pharmacology of lecular recognition: the effects of ligand and protein flexibility on molecular docking dopamine receptors. Pharmacol. Rev. 63, 182–217. https://doi.org/10.1124/pr.110. accuracy. J. Med. Chem. 47, 45–55. https://doi.org/10.1021/jm030209y. 002642. Evans, T.J., 2017. Endocrine disruption. In: Gupta, R.C. (Ed.), Reproductive and Benvenga, S., Guarneri, F., 2016. Conservation in the phylum of the local homology of Developmental Toxicology, second ed. Academic Press, Cambridge, pp. 1091–1110. apolipoproteins with the thyroid hormone plasma carriers. Rev. Endocr. Metab. https://doi.org/10.1016/B978-0-12-804239-7.00058-5. Disord. 17, 537–544. https://doi.org/10.1007/s11154-016-9379-7. Fang, L., Zhang, S., Chen, Z., Du, H., Zhu, Q., Dong, Z., Li, H., 2015. Risk assessment of Bernieri, T., Rodrigues, D., Barbosa, I.R., Ardenghi, P.G., da Silva, L.B., 2019. pesticide residues in dietary intake of celery in China. Regul. Toxicol. Pharmacol. 73, Occupational exposure to pesticides and thyroid function in Brazilian soybean 578–586. https://doi.org/10.1016/j.yrtph.2015.08.009. farmers. Chemosphere 218, 425–429. https://doi.org/10.1016/j.chemosphere.2018. Forsberg, N.D., Rodriguez-Proteau, R., Ma, L., Morré, J., Christensen, J.M., Maier, C.S., 11.124. Jenkins, J.J., Anderson, K.A., 2011. Organophosphorus pesticide degradation product Berto-Júnior, C., Santos-Silva, A.P., Ferreira, A.C.F., Graceli, J.B., de Carvalho, D.P., in vitro metabolic stability and time-course uptake and elimination in rats following Soares, P., Romeiro, N.C., Miranda-Alves, L., 2018. Unraveling molecular targets of oral and intravenous dosing. Xenobiotica 41, 422–429. https://doi.org/10.3109/ bisphenol A and S in the thyroid gland. Environ. Sci. Pollut. Res. 25, 26916–26926. 00498254.2010.550656. https://doi.org/10.1007/s11356-018-2419-y. Geng, X., Shao, H., Zhang, Z.H., Ng, J.C., Peng, C., 2015. Malathion-induced testicular Bhandari, G., Zomer, P., Atreya, K., Mol, H.G., Yang, X., Geissen, V., 2019. Pesticide toxicity is associated with spermatogenic apoptosis and alterations in testicular en- residues in Nepalese vegetables and potential health risks. Environ. Res. 172, zymes and hormone levels in male Wistar rats. Environ. Toxicol. Pharmacol. 39, 511–521. https://doi.org/10.1016/j.envres.2019.03.002. 659–667. https://doi.org/10.1016/j.etap.2015.01.010. Bhanot, R., Sangha, G.K., 2018. Effect of in utero and lactational exposure of triazophos Giulivo, M., de Alda, M.L., Capri, E., Barceló, D., 2016. Human exposure to endocrine on reproductive system functions in male offsprings, Rattus norvegicus. Drug Chem. disrupting compounds: their role in reproductive systems, metabolic syndrome and Toxicol. 1–7. https://doi.org/10.1080/01480545.2018.1457048. breast cancer. A review. Environ. Res. 151, 251–264. https://doi.org/10.1016/j. Bock, R., Their, W., 1976. Metabolism and fate of triazophos in rats. Pestic. Sci. 7, envres.2016.07.011. 307–314. https://doi.org/10.1002/ps.2780070316. Goldman, A.L., Bhasin, S., Wu, F.C., Krishna, M., Matsumoto, A.M., Jasuja, R., 2017. A Bolton, J.L., Hayward, C., Direk, N., Lewis, J.G., Hammond, G.L., Hill, L.A., Anderson, A., reappraisal of testosterone's binding in circulation: physiological and clinical im- Huffman, J., Wilson, J.F., Campbell, H., Rudan, I., Wright, A., Hastie, N., Wild,S.H., plications. Endocr. Rev. 38, 302–324. https://doi.org/10.1210/er.2017-00025. Velders, F.P., Hofman, A., Uitterlinden, A.G., Lahti, J., Räikkönen, K., Kajantie, E., Goto, T., 2019. A review of the studies on food-derived factors which regulate energy Widen, E., Palotie, A., Eriksson, J.G., Kaakinen, M., Järvelin, M.R., Timpson, N.J., metabolism via the modulation of lipid-sensing nuclear receptors. Biosci. Biotechnol. Davey Smith, G., Ring, S.M., Evans, D.M., St Pourcain, B., Tanaka, T., Milaneschi, Y., Biochem. 83, 579–588. https://doi.org/10.1080/09168451.2018.1559025. Bandinelli, S., Ferrucci, L., van der Harst, P., Rosmalen, J.G., Bakker, S.J., Verweij, N., Grimm, F.A., Lehmler, H.J., He, X., Robertson, L.W., Duffel, M.W., 2013. Sulfated me- Dullaart, R.P., Mahajan, A., Lindgren, C.M., Morris, A., Lind, L., Ingelsson, E., tabolites of polychlorinated biphenyls are high-affinity ligands for the thyroid hor- Anderson, L.N., Pennell, C.E., Lye, S.J., Matthews, S.G., Eriksson, J., Mellstrom, D., mone transport protein transthyretin. Environ. Health Perspect. 121, 657–662. Ohlsson, C., Price, J.F., Strachan, M.W., Reynolds, R.M., Tiemeier, H., Walker, B.R., https://doi.org/10.1289/ehp.1206198. CORtisol NETwork (CORNET) Consortium, 2014. Genome wide association identifies Gronemeyer, H., Gustafsson, J.Å., Laudet, V., 2004. Principles for modulation of the common variants at the SERPINA6/SERPINA1 locus influencing plasma cortisol and nuclear receptor superfamily. Nat. Rev. Drug Discov. 3, 950–964. https://doi.org/10. corticosteroid binding globulin. PLoS Genet. 10, e1004474. https://doi.org/10. 1038/nrd1551. 1371/journal.pgen.1004474. Grumetto, L., Barbato, F., Russo, G., 2019. Scrutinizing the interactions between bi- Browne, P., Noyes, P.D., Casey, W.M., Dix, D.J., 2017. Application of adverse outcome sphenol analogues and plasma proteins: insights from biomimetic liquid chromato- pathways to US EPA's endocrine disruptor screening program. Environ. Health graphy, molecular docking simulations and in silico predictions. Environ. Toxicol. Perspect. 125, 096001. https://doi.org/10.1289/EHP1304. Pharmacol. 68, 148–154. https://doi.org/10.1016/j.etap.2019.02.008. Browne, P., Casey, W.M., Dix, D.J., 2018. Use of high-throughput and computational Harvey, P.W., 2016. Adrenocortical endocrine disruption. J. Steroid Biochem. 155, approaches for endocrine pathway screening. In: Garcia-Reyero, N., Murphy, C. 199–206. https://doi.org/10.1016/j.jsbmb.2014.10.009. (Eds.), A Systems Biology Approach to Advancing Adverse Outcome Pathways for Harvey, P.W., Everett, D.J., Springall, C.J., 2007. Adrenal toxicology: a strategy for as- Risk Assessment. Springer, Cham, pp. 15–29. https://doi.org/10.1007/978-3-319- sessment of functional toxicity to the adrenal cortex and steroidogenesis. J. Appl. 66084-4_2. Toxicol. 27, 103–115. https://doi.org/10.1002/jat.1221. Burgdorf, T., Piersma, A.H., Landsiedel, R., Clewell, R., Kleinstreuer, N., Oelgeschläger, Hazarika, J., Ganguly, M., Mahanta, R., 2019. Molecular interactions of chlorpyrifos and M., Desprez, B., Kienhuis, A., Bos, P., de Vries, R., de Wit, L., Seidle, T., Scheel, J., its environmental degradation products with human sex hormone‐binding globulin: Schönfelder, G., van Benthem, J., Vinggaard, A.M., Eskes, C., Ezendam, J., 2019. an in silico study. J. Appl. Toxicol. 1–10. https://doi.org/10.1002/jat.3789. Workshop on the validation and regulatory acceptance of innovative 3R approaches He, D., Ye, X., Xiao, Y., Zhao, N., Long, J., Zhang, P., Fan, Y., Ding, S., Jin, X., Tian, C., Xu, in regulatory toxicology–Evolution versus revolution. Toxicol. In Vitro 59, 1–11. S., Ying, C., 2015. Dietary exposure to endocrine disrupting chemicals in me- https://doi.org/10.1016/j.tiv.2019.03.039. tropolitan population from China: a risk assessment based on probabilistic approach. Cavaliere, F., Cozzini, P., 2018. New in silico trends in food toxicology. Chem. Res. Chemosphere 139, 2–8. https://doi.org/10.1016/j.chemosphere.2015.05.036. Toxicol. 31, 992–993. https://doi.org/10.1021/acs.chemrestox.8b00133. Henretig, F.M., Kirk, M.A., McKay Jr., C.A., 2019. Hazardous chemical emergencies and Chebab, S., Mekircha, F., Leghouchi, E., 2017. Potential protective effect of Pistacia len- poisonings. N. Engl. J. Med. 380, 1638–1655. https://doi.org/10.1056/ tiscus oil against chlorpyrifos-induced hormonal changes and oxidative damage in NEJMra1504690. ovaries and thyroid of female rats. Biomed. Pharmacother. 96, 1310–1316. https:// Hill, K.L., Mortensen, Å.K., Teclechiel, D., Willmore, W.G., Sylte, I., Jenssen, B.M., doi.org/10.1016/j.biopha.2017.11.081. Letcher, R.J., 2018. In vitro and in silico competitive binding of brominated poly- Chen, C., Li, Y., Chen, M., Chen, Z., Qian, Y., 2009. Organophosphorus pesticide residues phenyl ether contaminants with human and gull thyroid hormone transport proteins. in milled rice (Oryza sativa) on the Chinesemarket and dietary risk assessment. Food Environ. Sci. Technol. 52, 1533–1541. https://doi.org/10.1021/acs.est.7b04617. Addit. Contam. A 26, 340–347. https://doi.org/10.1080/02652030802524516. Holden, A.J., Chen, L., Shaw, I.C., 2001. Thermal stability of organophosphorus pesticide Chen, Q., Tan, H., Yu, H., Shi, W., 2018. Activation of steroid hormone receptors: shed triazophos and its relevance in the assessment of risk to the consumer of triazophos light on the in silico evaluation of endocrine disrupting chemicals. Sci. Total Environ. residues in food. J. Agric. Food Chem. 49, 103–106. https://doi.org/10.1021/ 631–632, 27–39. https://doi.org/10.1016/j.scitotenv.2018.03.003. jf0002589. Chen, Q., Wang, X., Tan, H., Shi, W., Zhang, X., Wei, S., Giesy, J.P., Yu, H., 2019. Hong, S., She, Y., Cao, X., Wang, M., He, Y., Zheng, L., Wang, S., Abd El-Aty, A.M., Molecular initiating events of bisphenols on AR-mediated pathways provide guide- Hacimüftüoglu, A., Yan, M., Wang, J., 2019. A novel CdSe/ZnS quantum dots lines for in silico screening and design of substitute compounds. Environ. Sci. Technol. fluorescence assay based on molecularly imprinted sensitive membranes forde- Lett. 6, 205–210. https://doi.org/10.1021/acs.estlett.9b00073. termination of triazophos residues in cabbage and apple. Front. Chem. 7, 130. Cheurfa, M., Abdallah, H.H., Allem, R., Noui, A., Picot-Allain, C.M.N., Mahomoodally, F., https://doi.org/10.3389/fchem.2019.00130. 2019. Hypocholesterolaemic and antioxidant properties of Olea europaea L. leaves Jain, S., Ahmed, R.S., Arora, V.K., Banerjee, B.D., 2011. Biochemical and histopatholo- from Chlef province, Algeria using in vitro, in vivo and in silico approaches. Food gical studies to assess chronic toxicity of triazophos in blood, liver and brain tissue of Chem. Toxicol. 123, 98–105. https://doi.org/10.1016/j.fct.2018.10.002. rats. Pestic. Biochem. Physiol. 100, 182–186. https://doi.org/10.1016/j.pestbp.2011.

12 F.-W. Yang, et al. Food and Chemical Toxicology 133 (2019) 110759

03.008. sativa oil against reproductive toxicity, hormonal alterations, and oxidative damage Jain, S., Banerjee, B.D., Ahmed, R.S., Arora, V.K., Mediratta, P.K., 2013. Possible role of induced by chlorpyrifos in male rats. Toxicol. Ind. Health 32, 1266–1277. https:// oxidative stress and brain derived neurotrophic factor in triazophos induced cogni- doi.org/10.1177/0748233714554675. tive impairment in rats. Neurochem. Res. 38, 2136–2147. https://doi.org/10.1007/ OECD, 2018. Revised Guidance Document 150 on Standardised Test Guidelines for s11064-013-1122-0. Evaluating Chemicals for Endocrine Disruption, OECD Series on Testing and Jallouli, M., Bini Dhouib, I., Dhouib, H., Lasram, M., Gharbi, N., Fazaa, S., 2016. Assessment, No. 150. OECD Publishing, Paris. https://doi.org/10.1787/ Disruption of steroidogenesis after dimethoate exposure and efficacy of N-acet- 9789264304741-en. ylcysteine in rats: an old drug with new approaches. Environ. Sci. Pollut. Res. 23, Omoike, O.E., Lewis, R.C., Meeker, J.D., 2015. Association between urinary biomarkers of 7975–7984. https://doi.org/10.1007/s11356-016-6070-1. exposure to organophosphate insecticides and serum reproductive hormones in men Janssen, S.T., Janssen, O.E., 2017. Directional thyroid hormone distribution via the blood from NHANES 1999–2002. Reprod. Toxicol. 53, 99–104. https://doi.org/10.1016/j. stream to target sites. Mol. Cell. Endocrinol. 458, 16–21. https://doi.org/10.1016/j. reprotox.2015.04.005. mce.2017.02.037. Pan, Y., Mo, J., Wei, Y., Xie, Y., 2012. Determination of chlorpyrifos metabolite DEP in Jeong, J., Kim, H., Choi, J., 2019. In silico molecular docking and in vivo validation with green string bean using HPLC-MS/MS. Guangxi Sci. 19, 348–351. https://doi.org/10. caenorhabditis elegans to discover molecular initiating events in adverse outcome 13656/j.cnki.gxkx.2012.04.007. pathway framework: case study on endocrine-disrupting chemicals with estrogen and Pande, P., Fleck, S.C., Twaddle, N.C., Churchwell, M.I., Doerge, D.R., Teeguarden, J.G., androgen receptors. Int. J. Mol. Sci. 20, 1209. https://doi.org/10.3390/ 2019. Comparative estrogenicity of endogenous, environmental and dietary estrogens ijms20051209. in pregnant women II: total estrogenicity calculations accounting for competitive Joint FAO/WHO Meeting on Pesticide Residues (JMPR), 2002. Pesticide Residues in Food protein and receptor binding and potency. Food Chem. Toxicol. 125, 341–353. - 2002 - Joint FAO/WHO Meeting on Pesticide Residues. TRIAZOPHOS. http://www. https://doi.org/10.1016/j.fct.2018.12.013. inchem.org/documents/jmpr/jmpmono/2002pr14.htm. Panuwet, P., Ladva, C., Barr, D.B., Prapamontol, T., Meeker, J.D., D'Souza, P.E., John, E.M., Shaike, J.M., 2015. Chlorpyrifos: pollution and remediation. Environ. Chem. Maldonado, H., Ryan, P.B., Robson, M.G., 2018. Investigation of associations be- Lett. 13, 269–291. https://doi.org/10.1007/s10311-015-0513-7. tween exposures to pesticides and testosterone levels in Thai farmers. Arch. Environ. Kar, S., Sepúlveda, M.S., Roy, K., Leszczynski, J., 2017. Endocrine-disrupting activity of Occup. Health 73, 205–218. https://doi.org/10.1080/19338244.2017.1378606. per-and polyfluoroalkyl substances: exploring combined approaches of ligand and Peiris, D.C., Dhanushka, T., 2017. Low doses of chlorpyrifos interfere with spermato- structure based modeling. Chemosphere 184, 514–523. https://doi.org/10.1016/j. genesis of rats through reduction of sex hormones. Environ. Sci. Pollut. Res. 24, chemosphere.2017.06.024. 20859–20867. https://doi.org/10.1007/s11356-017-9617-x. Karplus, M., Levitt, M., Warshel, A., 2013. The Nobel Prize in Chemistry 2013Nobel Peng, W., Ding, F., Xie, Y., 2016. Biointeractions of CI Acid Red 2 and its structural Media AB. https://www.nobelprize.org/uploads/2018/06/press-103.pdf. analogues with transporter albumin: fluorescence, circular dichroism, and ligand Katsikantami, I., Colosio, C., Alegakis, A., Tzatzarakis, M.N., Vakonaki, E., Rizos, A.K., docking approaches. J. Photochem. Photobiol., B 154, 40–50. https://doi.org/10. Sarigiannis, D.A., Tsatsakis, A.M., 2019. Estimation of daily intake and risk assess- 1016/j.jphotobiol.2015.11.010. ment of organophosphorus pesticides based on biomonitoring data–the internal ex- Prasanth, G.K., Divya, L.M., Sadasivan, C., 2010. Bisphenol‐A can bind to human gluco- posure approach. Food Chem. Toxicol. 123, 57–71. https://doi.org/10.1016/j.fct. corticoid receptor as an agonist: an in silico study. J. Appl. Toxicol. 30, 769–774. 2018.10.047. https://doi.org/10.1002/jat.1570. Kawahata, I., Yamakuni, T., 2018. Imidacloprid, a neonicotinoid insecticide, facilitates Pytka, K., Podkowa, K., Rapacz, A., Podkowa, A., Żmudzka, E., Olczyk, A., Sapa, J., tyrosine hydroxylase transcription and phenylethanolamine N-methyltransferase Filipek, B., 2016. The role of serotonergic, adrenergic and dopaminergic receptors in mRNA expression to enhance catecholamine synthesis and its nicotine-evoked ele- antidepressant-like effect. Pharmacol. Rep. 68, 263–274. https://doi.org/10.1016/j. vation in PC12D cells. Toxicology 394, 84–92. https://doi.org/10.1016/j.tox.2017. pharep.2015.08.007. 12.004. Raees, K., Ishfaq, R., Ullah, A., Tahir, M.Z., Abbas, T., Tahir, H.M., Mukhtar, M.K., Arshad, Khan, M.F., Alam, M.M., Verma, G., Akhtar, W., Rizvi, M.A., Ali, A., Mymoona, A., M., Khan, S.Y., Ahmad, K.R., 2012. Histological and micrometric effects of diazinon Shaquiquzzaman, M., 2017. Molecular interactions of dioxins and DLCs with the exposure on adrenal medulla and cortex in mice. J. Appl. Anim. Res. 40, 267–272. ketosteroid receptors: an in silico risk assessment approach. Toxicol. Mech. Methods https://doi.org/10.1080/09712119.2012.672309. 27, 151–163. https://doi.org/10.1080/15376516.2016.1273423. Reemtsma, T., Lingott, J., Roegler, S., 2011. Determination of 14 monoalkyl phosphates, Kumari, D., John, S., 2019. Health risk assessment of pesticide residues in fruits and dialkyl phosphates and dialkyl thiophosphates by LC-MS/MS in human urinary vegetables from farms and markets of Western Indian Himalayan Region. samples. Sci. Total Environ. 409, 1990–1993. https://doi.org/10.1016/j.scitotenv. Chemosphere 224, 162–167. https://doi.org/10.1016/j.chemosphere.2019.02.091. 2011.01.032. Kvetnansky, R., Sabban, E.L., Palkovits, M., 2009. Catecholaminergic systems in stress: Ren, X.M., Zhang, Y.F., Guo, L.H., Qin, Z.F., Lv, Q.Y., Zhang, L.Y., 2015. structural and molecular genetic approaches. Physiol. Rev. 89, 535–606. https://doi. Structure–activity relations in binding of perfluoroalkyl compounds to human thyroid org/10.1152/physrev.00042.2006. hormone T3 receptor. Arch. Toxicol. 89, 233–242. https://doi.org/10.1007/s00204- Kvetnansky, R., Lu, X., Ziegler, M.G., 2013. Stress-triggered changes in peripheral ca- 014-1258-y. techolaminergic systems. In: In: Eiden, L.E. (Ed.), A New Era of Catecholamines in the Ren, X.M., Qin, W.P., Cao, L.Y., Zhang, J., Yang, Y., Wan, B., Guo, L.H., 2016. Binding Laboratory and Clinic, vol. 68. Academic Press, pp. 359–397. https://doi.org/10. interactions of perfluoroalkyl substances with thyroid hormone transport proteins 1016/B978-0-12-411512-5.00017-8. and potential toxicological implications. Toxicology 366, 32–42. https://doi.org/10. La Perle, K.M.D., Dintzis, S.M., 2018. Endocrine system. In: Treuting, P.M., Dintzis, S.M., 1016/j.tox.2016.08.011. Montine, K.S. (Eds.), Comparative Anatomy and Histology, second ed. Academic Ribeiro Filho, H.V., Tambones, I.L., Dias, M.M.G., Videira, N.B., Bruder, M., Amato, A.A., Press, Cambridge, pp. 251–273. https://doi.org/10.1016/B978-0-12-802900-8. Figueira, A.C.M., 2019. Modulation of nuclear receptor function: targeting the pro- 00015-4. tein-DNA interface. Mol. Cell. Endocrinol. 484, 1–14. https://doi.org/10.1016/j.mce. Le Maire, A., Bourguet, W., Balaguer, P., 2010. A structural view of nuclear hormone 2019.01.023. receptor: endocrine disruptor interactions. Cell. Mol. Life Sci. 67, 1219–1237. Rosol, T.J., DeLellis, R.A., Harvey, P.W., Sutcliffe, C., 2013. Endocrine system. In: third https://doi.org/10.1007/s00018-009-0249-2. ed. In: Haschek, W.M., Rousseaux, C.G., Wallig, M.A. (Eds.), Haschek and Rousseaux's Li, F., Xie, Q., Li, X., Li, N., Chi, P., Chen, J., Wang, Z., Hao, C., 2010. Hormone activity of Handbook of Toxicologic Pathology, vol. III. Academic Press, Cambridge, pp. hydroxylated polybrominated diphenyl ethers on human thyroid receptor-β: in vitro 2391–2492. https://doi.org/10.1016/B978-0-12-415759-0.00058-3. and in silico investigations. Environ. Health Perspect. 118, 602–606. https://doi.org/ Ruiz, P., Sack, A., Wampole, M., Bobst, S., Vracko, M., 2017. Integration of in silico 10.1289/ehp.0901457. methods and computational systems biology to explore endocrine-disrupting che- Li, P., Zeng, X.-Y., Cui, J.-T., Zhao, L.-J., Yu, Z.-Q., 2017. Determination of seven urinary mical binding with nuclear hormone receptors. Chemosphere 178, 99–109. https:// metabolites of organophosphate esters using liquid chromatography-tandem mass doi.org/10.1016/j.chemosphere.2017.03.026. spectrometry. Chin. J. Anal. Chem. 45, 1648–1654. https://doi.org/10.1016/S1872- Sarath Josh, M.K., Pradeep, S., Vijayalekshmy Amma, K.S., Sudha Devi, R., Balachandran, 2040(17)61048-X. S., Sreejith, M.N., Benjamin, S., 2016. Human ketosteroid receptors interact with Liu, H., Yang, X., Lu, R., 2016. Development of classification model and QSAR model for hazardous phthalate plasticizers and their metabolites: an in silico study. J. Appl. predicting binding affinity of endocrine disrupting chemicals to human sex hormone- Toxicol. 36, 836–843. https://doi.org/10.1002/jat.3221. binding globulin. Chemosphere 156, 1–7. https://doi.org/10.1016/j.chemosphere. Schwalbe-Fehl, M., Schmidt, E., 1986. Hoe 002960-14-C, Triazophos, Comparative 2016.04.077. Metabolism Study in Rats and Dogs. Unpublished report No. CM048/85 from Liu, J.J., Hezghia, A., Shaikh, S.R., Cenido, J.F., Stark, R.E., Mann, J.J., Sublette, M.E., Hoechst AG, Frankfurt am Main, Germany, 4 February 1986. Aventis document 2018. Regulation of monoamine transporters and receptors by lipid microdomains: A32754. Submitted to WHO by Aventis CropScience, Frankfurt am Main, Germany. implications for depression. Neuropsychopharmacology 43, 2165–2179. https://doi. Selvaraj, C., Sakkiah, S., Tong, W., Hong, H., 2018. Molecular dynamics simulations and org/10.1038/s41386-018-0133-6. applications in computational toxicology and nanotoxicology. Food Chem. Toxicol. Lu, L., Zhan, T., Ma, M., Xu, C., Wang, J., Zhang, C., Liu, W., Zhuang, S., 2018. Thyroid 112, 495–506. https://doi.org/10.1016/j.fct.2017.08.028. disruption by bisphenol S analogues via thyroid hormone receptor β: in vitro, in vivo, Sharma, D., Sangha, G.K., 2014. Triazophos induced oxidative stress and histomorpho- and molecular dynamics simulation study. Environ. Sci. Technol. 52, 6617–6625. logical changes in liver and kidney of female albino rats. Pestic. Biochem. Physiol. https://doi.org/10.1021/acs.est.8b00776. 110, 71–80. https://doi.org/10.1016/j.pestbp.2014.03.003. Mimoto, M.S., Nadal, A., Sargis, R.M., 2017. Polluted pathways: mechanisms of metabolic Sharma, D., Sangha, G.K., Khera, K.S., 2015a. Triazophos-induced oxidative stress and disruption by endocrine disrupting chemicals. Curr. Environ. Health Rep. 4 (2), histomorphological changes in ovary of female Wistar rats. Pestic. Biochem. Physiol. 208–222. https://doi.org/10.1007/s40572-017-0137-0. 117, 9–18. https://doi.org/10.1016/j.pestbp.2014.09.004. Mohammad, J., Scanni, R., Bestmann, L., Hulter, H.N., Krapf, R., 2018. A controlled in- Sharma, D., Sangha, G.K., Khera, K.S., 2015b. Effect of preconceptional exposure of crease in dietary phosphate elevates BP in healthy human subjects. J. Am. Soc. triazophos formulation on fertility and reproductive performance of female Wistar Nephrol. 29, 2089–2098. https://doi.org/10.1681/ASN.2017121254. rats, Rattus norvegicus. P. Natl. A. Sci. India B 85, 987–992. https://doi.org/10.1007/ Mosbah, R., Yousef, M.I., Maranghi, F., Mantovani, A., 2016. Protective role of Nigella s40011-014-0460-2.

13 F.-W. Yang, et al. Food and Chemical Toxicology 133 (2019) 110759

Sharma, S., Ahmad, S., Khan, M.F., Parvez, S., Raisuddin, S., 2018. In silico molecular mechanisms of organophosphate insecticides. Chin. J. Pesticide Sci. 17, 1–14. interaction of bisphenol analogues with human nuclear receptors reveals their https://doi.org/10.3969/j.issn.1008-7303.015.01.01. stronger affinity vs. classical bisphenol A. Toxicol. Mech. Methods 28, 660–669. Wang, X., Zhang, X., Xia, P., Zhang, J., Wang, Y., Zhang, R., Giesy, J.P., Shi, W., Yu, H., https://doi.org/10.1080/15376516.2018.1491663. 2017a. A high-throughput, computational system to predict if environmental con- Sheikh, I.A., Beg, M.A., 2017. Endocrine disruption: In silico interactions between taminants can bind to human nuclear receptors. Sci. Total Environ. 576, 609–616. phthalate plasticizers and corticosteroid binding globulin. J. Appl. Toxicol. 37, https://doi.org/10.1016/j.scitotenv.2016.10.093. 1471–1480. https://doi.org/10.1002/jat.3497. Wang, Y., Chen, L., Wang, C., Hum, Y., Gao, Y., Zhou, Y., Shi, R., Zhang, Y., Kamijima, M., Sheikh, I.A., Beg, M.A., 2019. Structural characterization of potential endocrine dis- Ueyama, J., Tian, Y., 2017b. Association between organophosphate pesticide ex- rupting activity of alternate plasticizers di-(2-ethylhexyl) adipate (DEHA), acetyl posure and thyroid hormones in pregnant women. Epidemiology 28, S35–S40. tributyl citrate (ATBC) and 2, 2, 4-trimethyl 1, 3-pentanediol diisobutyrate (TPIB) https://doi.org/10.1097/EDE.0000000000000721. with human sex hormone-binding globulin. Reprod. Toxicol. 83, 46–53. https://doi. Weikum, E.R., Knuesel, M.T., Ortlund, E.A., Yamamoto, K.R., 2017. Glucocorticoid re- org/10.1016/j.reprotox.2018.11.003. ceptor control of transcription: precision and plasticity via allostery. Nat. Rev. Mol. Sheikh, I.A., Abu-Elmagd, M., Turki, R.F., Damanhouri, G.A., Beg, M.A., Al-Qahtani, M., Cell Biol. 18, 159–174. https://doi.org/10.1038/nrm.2016.152. 2016a. Endocrine disruption: In silico perspectives of interactions of di-(2-ethylhexyl) Weikum, E.R., Liu, X., Ortlund, E.A., 2018. The nuclear receptor superfamily: a structural phthalate and its five major metabolites with progesterone receptor. BMC Struct. perspective. Protein Sci. 27, 1876–1892. https://doi.org/10.1002/pro.3496. Biol. 16, 16. https://doi.org/10.1186/s12900-016-0066-4. World Health Organization (WHO), 2010. The WHO Recommended Classification of Sheikh, I.A., Turki, R.F., Abuzenadah, A.M., Damanhouri, G.A., Beg, M.A., 2016b. Pesticides by Hazard and Guidelines to Classification 2009. WHO, pp. 21–23. Endocrine disruption: computational perspectives on human sex hormone-binding Wu, G., Robertson, D.H., Brooks III, C.L., Vieth, M., 2003. Detailed analysis of grid-based globulin and phthalate plasticizers. PLoS One 11, e0151444. https://doi.org/10. molecular docking: a case study of CDOCKER - a CHARMm-based MD docking al- 1371/journal.pone.0151444. gorithm. J. Comput. Chem. 24, 1549. https://doi.org/10.1002/jcc.10306. Sheikh, I.A., Yasir, M., Abu-Elmagd, M., Dar, T.A., Abuzenadah, A.M., Damanhouri, G.A., Wu, S., Li, X., Liu, X., Yang, G., An, X., Wang, Q., Wang, Y., 2018. Joint toxic effects of Al-Qahtani, M., Beg, M.A., 2016c. Human sex hormone-binding globulin as a po- triazophos and imidacloprid on zebrafish (Danio rerio). Environ. Pollut. 235, tential target of alternate plasticizers: an in silico study. BMC Struct. Biol. 16, 15. 470–481. https://doi.org/10.1016/j.envpol.2017.12.120. https://doi.org/10.1186/s12900-016-0067-3. Xin, Y., Ren, X.M., Ruan, T., Li, C.H., Guo, L.H., Jiang, G., 2018. Chlorinated poly- Shen, W.J., Azhar, S., Kraemer, F.B., 2016. ACTH regulation of adrenal SR-B1. Front. fluoroalkylether sulfonates exhibit similar binding potency and activity to thyroid Endocrinol. 7, 42. https://doi.org/10.3389/fendo.2016.00042. hormone transport proteins and nuclear receptors as perfluorooctanesulfonate. Shomar, B., Al-Saad, K., Nriagu, J., 2014. Mishandling and exposure of farm workers in Environ. Sci. Technol. 52, 9412–9418. https://doi.org/10.1021/acs.est.8b01494. Qatar to organophosphate pesticides. Environ. Res. 133, 312–320. https://doi.org/ Xiong, J., Tian, L., Qiu, Y., Sun, D., Zhang, H., Wu, M., Wang, J., 2017. Evaluation on the 10.1016/j.envres.2014.06.010. thyroid disrupting mechanism of malathion in Fischer rat thyroid follicular cell line Shrestha, S., Parks, C.G., Goldner, W.S., Kamel, F., Umbach, D.M., Ward, M.H., Lerro, FRTL-5. Drug Chem. Toxicol. 1–8. https://doi.org/10.1080/01480545.2017. C.C., Koutros, S., Hofmann, J.N., Freeman, L.E.B., Sandler, D.P., 2018. Pesticide use 1397162. and incident hypothyroidism in pesticide applicators in the agricultural health study. Yuriev, E., Holien, J., Ramsland, P.A., 2015. Improvements, trends, and new ideas in Environ. Health Perspect. 126, 097008. https://doi.org/10.1289/EHP3194. molecular docking: 2012–2013 in review. J. Mol. Recognit. 28, 581–604. https://doi. Sudakin, D.L., Stone, D.L., 2011. Dialkyl phosphates as biomarkers of organophosphates: org/10.1002/jmr.2471. the current divide between epidemiology and clinical toxicology. Clin. Toxicol. 49, Yusa, V., Millet, M., Coscolla, C., Pardo, O., Roca, M., 2015. Occurrence of biomarkers of 771–781. https://doi.org/10.3109/15563650.2011.624101. pesticide exposure in non-invasive human specimens. Chemosphere 139, 91–108. Sun, Y., Gong, X., Lin, W., Liu, Y., Wang, Y., Wu, M., Kannan, K., Ma, J., 2018. Metabolites https://doi.org/10.1016/j.chemosphere.2015.05.082. of organophosphate ester flame retardants in urine from Shanghai, China. Environ. Zengin, G., Abdallah, H.H., Dogan, A., Mollica, A., Aumeeruddy-Elalfi, Z., Mahomoodally, Res. 164, 507–515. https://doi.org/10.1016/j.envres.2018.03.031. M.F., 2018. Phenolic components and assessment of biological properties of Sun, L., Yang, H., Cai, Y., Li, W., Liu, G., Tang, Y., 2019. In silico prediction of endocrine Tchihatchewia isatidea Boiss. extracts: docking and functional approaches for de- disrupting chemicals using single-label and multi-label models. J. Chem. Inf. Model. signing novel products. Food Chem. Toxicol. 111, 423–431. https://doi.org/10. 59, 973–982. https://doi.org/10.1021/acs.jcim.8b00551. 1016/j.fct.2017.11.055. Targovnik, H.M., Citterio, C.E., Rivolta, C.M., 2017. Iodide handling disorders (NIS, TPO, Zhang, X., Driver, J.H., Li, Y., Ross, J.H., Krieger, R.I., 2008. Dialkylphosphates (DAPs) in TG, IYD). Best Pract. Res. Cli. En. 31, 195–212. https://doi.org/10.1016/j.beem. fruits and vegetables may confound biomonitoring in organophosphorus insecticide 2017.03.006. exposure and risk assessment. J. Agric. Food Chem. 56, 10638–10645. https://doi. Thomas, S.R., McTamney, P.M., Adler, J.M., LaRonde-LeBlanc, N., Rokita, S.E., 2009. org/10.1021/jf8018084. Crystal structure of iodotyrosine deiodinase, a novel flavoprotein responsible for Zhang, Z., Yuan, Y., Zheng, W., Sun, C., Yang, G., Wang, Q., 2011. Dietary intake and its iodide salvage in thyroid glands. J. Biol. Chem. 284, 19659–19667. https://doi.org/ risk assessment of triazophos residue. Chin. J. Pesticide Sci. 13, 485–495. https://doi. 10.1074/jbc.M109.013458. org/10.3969/j.issn.1008-7303.2011.05.09. Timchalk, C., Busby, A., Campbell, J.A., Needham, L.L., Barr, D.B., 2007. Comparative Zhang, J., Kamstra, J.H., Ghorbanzadeh, M., Weiss, J.M., Hamers, T., Andersson, P.L., pharmacokinetics of the organophosphorus insecticide chlorpyrifos and its major 2015. In silico approach to identify potential thyroid hormone disruptors among metabolites diethylphosphate, diethylthiophosphate and 3, 5, 6-trichloro-2-pyridinol currently known dust contaminants and their metabolites. Environ. Sci. Technol. 49, in the rat. Toxicology 237, 145–157. https://doi.org/10.1016/j.tox.2007.05.007. 10099–10107. https://doi.org/10.1021/acs.est.5b01742. Ueyama, J., Harada, K.H., Koizumi, A., Sugiura, Y., Kondo, T., Saito, I., Kamijima, M., Zhang, J., Begum, A., Brännström, K., Grundström, C., Iakovleva, I., Olofsson, A., Sauer- 2015. Temporal levels of urinary neonicotinoid and dialkylphosphate concentrations Eriksson, A.E., Andersson, P.L., 2016a. Structure-based virtual screening protocol for in Japanese women between 1994 and 2011. Environ. Sci. Technol. 49, in silico identification of potential thyroid disrupting chemicals targeting transthyr- 14522–14528. https://doi.org/10.1021/acs.est.5b03062. etin. Environ. Sci. Technol. 50, 11984–11993. https://doi.org/10.1021/acs.est. Usman, A., Ahmad, M., 2019. Computational study suggesting reconsideration of BPA 6b02771. analogues based on their endocrine disrupting potential estimated by binding affi- Zhang, J., Li, Y., Gupta, A.A., Nam, K., Andersson, P.L., 2016b. Identification and mo- nities to nuclear receptors. Ecotoxicol. Environ. Saf. 171, 154–161. https://doi.org/ lecular interaction studies of thyroid hormone receptor disruptors among household 10.1016/j.ecoenv.2018.12.071. dust contaminants. Chem. Res. Toxicol. 29, 1345–1354. https://doi.org/10.1021/ Verma, G., Khan, M.F., Shaquiquzzaman, M., Akhtar, W., Akhter, M., Hasan, S.M., Alam, acs.chemrestox.6b00171. M.M., 2017. Molecular interactions of dioxins and DLCs with the xenosensors (PXR Zhang, J., Zhang, J., Liu, R., Gan, J., Liu, J., Liu, W., 2016c. Endocrine-disrupting effects and CAR): an in silico risk assessment approach. J. Mol. Recognit. 30, e2651. https:// of pesticides through interference with human glucocorticoid receptor. Environ. Sci. doi.org/10.1002/jmr.2651. Technol. 50, 435–443. https://doi.org/10.1021/acs.est.5b03731. Vuorinen, A., Odermatt, A., Schuster, D., 2015. Reprint of “In silico methods in the dis- Zhang, J., Grundström, C., Brannstrom, K., Iakovleva, I., Lindberg, M., Olofsson, A., covery of endocrine disrupting chemicals”. J. Steroid Biochem. 153, 93–101. https:// Andersson, P.L., Sauer-Eriksson, A.E., 2018a. Interspecies variation between fish and doi.org/10.1016/j.jsbmb.2015.08.015. human transthyretins in their binding of thyroid-disrupting chemicals. Environ. Sci. Wallig, M.A., 2018. Endocrine system. In: Wallig, M.A., Haschek, W.M., Rousseaux, C.G., Technol. 52, 11865–11874. https://doi.org/10.1021/acs.est.8b03581. Bolon, B. (Eds.), Fundamentals of Toxicologic Pathology, third ed. Academic Press, Zhang, J., Li, T., Wang, T., Yuan, C., Zhong, S., Guan, T., Li, Z., Wang, Y., Yu, H., Luo, Q., Cambridge, pp. 565–624. https://doi.org/10.1016/B978-0-12-809841-7.00020-4. Wang, Y., Zhang, T., 2018b. Estrogenicity of halogenated bisphenol A: in vitro and in Wang, X.X., Zhou, S.L., Ding, X.F., Zhu, G.N., Guo, J.F., 2010. Effect of triazophos, fipronil silico investigations. Arch. Toxicol. 92, 1215–1223. https://doi.org/10.1007/s00204- and their mixture on miRNA expression in adult zebrafish. J. Environ. Sci. Health B 017-2127-2. 45, 648–657. https://doi.org/10.1080/03601234.2010.502435. Zhang, T., Zhong, S., Li, T., Zhang, J., 2018c. Saponins as modulators of nuclear receptors. Wang, Z., Kang, Z., Shi, X., Gao, X., 2015. Research progresses on the metabolic Crit. Rev. Food Sci. 1–14. https://doi.org/10.1080/10408398.2018.1514580.